Sulfonylurea-responsive repressor proteins

ABSTRACT

Compositions and methods relating to the use of sulfonylurea-responsive repressors are provided. Compositions include polypeptides that specifically bind to an operator, wherein the specific binding is regulated by a sulfonylurea compound. Compositions also include polynucleotides encoding the polypeptides as well as constructs, vectors, prokaryotic and eukaryotic cells, and eukaryotic organisms including plants and seeds comprising the polynucleotide, and/or produced by the methods. Also provided are methods to provide a sulfonylurea-responsive repressor to a cell or organism, and to regulate expression of a polynucleotide of interest in a cell or organism, including a plant or plant cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.12/603,739 filed Oct. 22, 2009, now U.S. Pat. No. 8,257,956 which claimsthe benefit of U.S. Application Ser. No. 61/108,917 filed Oct. 28, 2008,which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the field of molecular biology, moreparticularly to the regulation of gene expression.

BACKGROUND

The tetracycline operon system, comprising repressor and operatorelements, was originally isolated from bacteria. The operon system istightly controlled by the presence of tetracycline, and self-regulatesthe level of expression of tetA and tetR genes. The product of tetAremoves tetracycline from the cell. The product of tetR is the repressorprotein that binds to the operator elements with a K_(d) of about 10 pMin the absence of tetracycline, thereby blocking expression or tetA andtetR.

This system has been modified to control expression of otherpolynucleotides of interest, and/or for use in other organisms, mainlyfor use in animal systems. Tet operon based systems have had limited usein plants, at least partially due to problems with the inducers whichare typically antibiotic compounds, and sensitive to light.

There is a need to regulate expression of sequences of interest inorganisms, compositions and methods to tightly regulate expression inresponse to sulfonylurea compounds are provided.

SUMMARY

Compositions and methods relating to the use of sulfonylurea-responsiverepressors are provided. Compositions include polypeptides thatspecifically bind to an operator, wherein the specific binding isregulated by a sulfonylurea compound. Compositions also includepolynucleotides encoding the polypeptides as well as constructs,vectors, prokaryotic and eukaryotic cells and eukaryotic organismsincluding plants and seeds comprising the polynucleotide and/or producedby the methods. Also provided are methods to provide asulfonylurea-responsive repressor to a cell or organism and to regulateexpression of a polynucleotide of interest in a cell or organism,including a plant or plant cell.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Docking of tetracycline-Mg++ and the sulfonylurea compoundHarmony® (thifensulfuron-methyl; Ts) into the binding pocket of class DTetR based on the crystal structure 1DU7 from the Protein Databank(PDB).

FIG. 2. Vector map of an exemplary E. coli-based tetR expression vector,pVER7314. The replicon backbone is based on that of pBR322. The TetRligand binding domain (LBD) is encoded flanked by SacI and AscI sites.KMsp172 and KMsp173 represent binding sites for the primers used for DNAsequencing of inserted tetR genes. rrnB T1 T2 is a strongtranscriptional terminator to inhibit run around transcription andunregulated tetR expression.

FIG. 3. Response of library 1 hits to 20 μg/ml thifensulfuron-methyl(Ts). E. coli KM3 cells harboring putative tetR hits L1-1 through L1-20or wt tetR were replica plated onto M9 assay medium +/−20 μg/ml Ts, thenincubated at 30° C. until blue/white colony discrimination was evident.At this time colonies were imaged and relative β-galactosidase activitydetermined based on degree of blue colony color.

FIG. 4. Relative β-galactosidase activities of 45 putative library L4hits against 0, 0.2 and 1.0 ppm ethametsulfuron (Es). Induced activitywas measured using 5 μl of perforated whole cell mixture, and backgroundactivity was measured using 25 μl perforated cell mixture so thatdetectable activity could be measured in the same time frame for alltreatments. Background activity values were multiplied by 10 in order tobring them into the display range of the graph. The right hand side ofthe graph contains the controls, wild type TetR and 1^(st) round hitL1-9.

FIG. 5. β-galactosidase induction in L7 hits with ethametsulfuron. Tophits from the L7 library were re-arrayed and tested in 96-well cultureformat for relative induction by 0.02 μg/ml and 0.2 μg/ml inducer (Es),and for background activity in the absence of inducer. Induced activitywas assayed using 5 μl of perforated cell mixture, whereas 25 μl ofcells was used to detect background activity. This allowed alldetectable activities to be measured in the same time frame for alltreatments. Background activity values were multiplied by ten to bringthem into the range of the graph. The latter part of the graph shows thecontrols: 2nd round hits L4-89 and L4-120, and wt TetR(B) withethametsulfuron; and wt TetR with 0.4 μM atc as cognate inducer forcomparison (diagonally striped bar). Well ID's indicated with slantedtext refer to that of the assay re-array whereas original clone ID's areindicated below in horizontal text.

FIG. 6. Ethametsulfuron dose response of two EsR variants determined bytransient expression in Nicotiana benthamiana leaves. Black barsrepresent wt TetR, grey bars represent EsR hit A11, and white barsrepresent EsR hit D01. The striped bar represents a no repressor controlwhich indicates the maximum level of reporter expression in the assay.

FIG. 7. DNA binding to tetOp in the absence or presence of ligand. Fivepmol TetO or control DNA was mixed with the indicated amounts ofrepressor protein and inducer in complex buffer containing 20 mMTris-HCl (pH8.0) and 10 mM EDTA.

FIG. 8. Structures of exemplary registered sulfonylurea compounds.

FIG. 9. Summary of source diversity, library design, and hit diversityand population bias for several generations of sulfonylurea repressorshuffling libraries. A dash (“-”) indicates no amino acid diversityintroduced at that position in that library. An X indicates that thelibrary oligos were designed to introduce complete amino acid diversity(any of 20 amino acids) at that position in that library. Residues inbold indicate bias during selection with larger font size indicating agreater degree of bias in the selected population. Residues inparentheses indicate selected mutations. The phylogenetic diversity poolis derived from a broad family of 34 tetracycline repressor sequences.

FIG. 10. Sulfonylurea depression of fluorescent reporter in maize callus(A) or plants (B).

FIG. 11. β-galactosidase induction in exemplary L13 hits withethametsulfuron.

DETAILED DESCRIPTION

Chemically regulated expression tools have proven valuable for studyinggene function and regulation in many biological systems. These systemsallow testing for the effect of expression of any gene of interest in aculture system or whole organism when the transgene cannot bespecifically regulated, or continuously expressed due to negativeconsequences. These systems essentially provide the opportunity to do“pulse” or “pulse-chase” gene expression testing. A chemicalswitch-mediated expression system allows testing of genomic, proteomic,and/or metabolomic responses immediately following activation of thetarget gene. These types of tests cannot be done with constitutive,developmental, or tissue-specific expression systems. Chemical switchtechnologies may also provide a means for gene therapy.

Chemical switch systems can be commercially applied, such as inagricultural biotechnology. For agricultural purposes it is desired tobe able to control the expression and/or genetic flow of transgenes inthe environment, such as herbicide resistance genes, especially in caseswhere weedy relatives of the target crop exist. In addition, having afamily of viable chemical switch mechanisms would enable trait inventorymanagement from a single transgenic crop, for example, one productionline could be used to deliver selected traits on customer demand viaspecific chemical activation. Additionally, hybrid seed production couldbe streamlined by using chemical control of hybrid maintenance.

The Tet repressor (TetR) based genetic switch system widely used inanimal systems has had limited use in plant genetic systems, due in partto problems with the activator ligands. TetR has been redesigned torecognize commercially used sulfonylurea chemistry instead oftetracycline compounds, while retaining the ability to specifically bindtetracycline operator sequences. This was accomplished by modifying theTet repressor ligand binding domain using rational protein modeling andDNA shuffling to recognize commercially used sulfonylurea compounds.Initial TetR shuffling and screening using a sensitive in vivoβ-galactosidase assay led to specific recognition of the herbicideHarmony® (thifensulfuron-methyl) at 20 ppm in the growth medium, andloss of recognition of tetracycline. Upon testing with othersulfonylurea compounds, many of the hits reactive to Harmony® alsoresponded to other SU compounds. In some cases, the hits had even betterreactivity to related herbicides chlorsulfuron and ethametsulfuron (2ppm). Further rounds of shuffling and screening of the TetR derivativesled to TetR variants that react robustly to 0.2 ppm chlorsulfuron and0.02 ppm ethametsulfuron as measured using in vivo induction assays inE. coli. Top performing ethametsulfuron responsive SuR variants (EsRs)show induction capacity nearly equal to that of wild type class B TetRinduction by anhydrotetracycline (atc) using similar inducerconcentrations. These SuR molecules have no reactivity to tetracyclines,and wild type TetR(B) (SEQ ID NO: 2) has no reactivity to thesulfonylureas.

Compositions and methods relating to the use of sulfonylurea-responsiverepressors are provided. Sulfonylurea-responsive repressors (SuRs)include any repressor polypeptide whose binding to an operator sequenceis controlled by a ligand comprising a sulfonylurea compound. In someexamples, the repressor binds specifically to the operator in theabsence of a sulfonylurea ligand. In some examples, the repressor bindsspecifically to the operator in the presence of a sulfonylurea ligand.Repressors that bind to an operator in the presence of the ligand aresometimes called a reverse repressor. In some examples compositionsinclude SuR polypeptides that specifically bind to a tetracyclineoperator, wherein the specific binding is regulated by a sulfonylureacompound. In some examples compositions include an isolated sulfonylurearepressor (SuR) polypeptide comprising at least one amino acidsubstitution to a wild type tetracycline repressor protein ligandbinding domain wherein the SuR polypeptide, or a multimer thereof,specifically binds to a polynucleotide comprising an operator sequence,wherein repressor-operator binding is regulated by the absence orpresence of a sulfonylurea compound. In some examples compositionsincluded isolated sulfonylurea repressors comprising a ligand bindingdomain comprising at least one amino acid substitution to a wild typetetracycline repressor protein ligand binding domain fused to aheterologous operator DNA binding domain which specifically binds to apolynucleotide comprising the operator sequence or derivative thereof,wherein repressor-operator binding is regulated by the absence orpresence of a sulfonylurea compound. Any operator DNA binding domain canbe used, including but not limited to an operator DNA binding domainfrom repressors included tet, lac, trp, phd, arg, LexA, phiCh1repressor, lambda C1 and Cro repressors, phage X repressor, MetJ, phir1trro, phi434 C1 and Cro repressors, RafR, gal, ebg, uxuR, exuR, ROS,SinR, PurR, FruR, P22 C2, TetC, AcrR, Betl, Bm3R1, EnvR, QacR, MtrR,TcmR, Ttk, YbiH, YhgD, and mu Ner, or DNA binding domains in Interprofamilies including but not limited to IPR001647, IPR010982, andIPR011991.

In some examples compositions include an isolated sulfonylurea repressor(SuR) polypeptides comprising at least one amino acid substitution to awild type tetracycline repressor protein wherein the SuR polypeptide, ora multimer thereof, specifically binds to a polynucleotide comprising atetracycline operator sequence, wherein repressor-operator binding isregulated by the absence or presence of a sulfonylurea compound.

Wild type repressors include tetracycline class A, B, C, D, E, G, H, Jand Z repressors. An example of the TetR(A) class is found on the Tn1721transposon and deposited under GenBank accession X61307, crossreferencedunder gi48198, with encoded protein accession CAA43639, crossreferencedunder gi48195 and UniProt accession Q56321. An example of the TetR(B)class is found on the Tn10 transposon and deposited under GenBankaccession X00694, crossreferenced under gi43052, with encoded proteinaccession CAA25291, crossreferenced under gi43052 and UniProt accessionP04483. An example of the TetR(C) class is found on the pSC101 plasmidand deposited under GenBank Accession M36272, crossreferenced undergi150945, with encoded protein accession AAA25677, crossreferenced undergi150946. An example of the TetR(D) class is found in Salmonella ordonezand deposited under GenBank Accession X65876, crossreferenced undergi49073, with encoded protein accession CAA46707, crossreferenced undergi49075 and UniProt accessions P0ACT5 and P09164. An example of theTetR(E) class was isolated from E. coli transposon Tn10 and depositedunder GenBank Accession M34933, crossreferenced under gi155019, withencoded protein accession AAA98409, crossreferenced under gi155020. Anexample of the TetR(G) class was isolated from Vibrio anguillarium anddeposited under GenBank Accession S52438, crossreferenced undergi262928, with encoded protein accession AAB24797, crossreferenced undergi262929. An example of the TetR(H) class is found on plasmid pMV111isolated from Pasteurella multocida and deposited under GenBankAccession U00792, crossreferenced under gi392871, with encoded proteinaccession AAC43249, crossreferenced under gi392872. An example of theTetR(J) class was isolated from Proteus mirabilis and deposited underGenBank Accession AF038993, crossreferenced under gi4104704, withencoded protein accession AAD12754, crossreferenced under gi4104706. Anexample of the TetR(Z) class was found on plasmid pAGI isolated fromCorynebacterium glutamicum and deposited under GenBank AccessionAF121000, crossreferenced under gi4583389, with encoded proteinaccession AAD25064, crossreferenced under gi4583390. In some examplesthe wild type tetracycline repressor is a class B tetracycline repressorprotein. In some examples the wild type tetracycline repressor is aclass D tetracycline repressor protein.

In some examples the sulfonylurea repressor (SuR) polypeptides comprisean amino acid substitution in the ligand binding domain of a wild typetetracycline repressor protein. In class B and D wild type TetRproteins, amino acid residues 6-52 represent the DNA binding domain. Theremainder of the protein is involved in ligand binding and subsequentallosteric modification. For class B TetR residues 53-207 represent theligand binding domain, while residues 53-218 comprise the ligand bindingdomain for the class D TetR. In some examples the SuR polypeptidescomprise an amino acid substitution in the ligand binding domain of awild type TetR(B) protein. In some examples the SuR polypeptidescomprise an amino acid substitution in the ligand binding domain of awild type TetR(B) protein of SEQ ID NO: 1.

In some examples the isolated SuR polypeptides comprise an amino acid,or any combination of amino acids, corresponding to equivalent aminoacid positions selected from the amino acid diversity shown in FIG. 9,wherein the amino acid residue position shown in FIG. 9 corresponds tothe amino acid numbering of a wild type TetR(B). In some examples theisolated SuR polypeptides comprise a ligand binding domain comprising atleast 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% of the amino acid residues shown in FIG. 9, wherein theamino acid residue position corresponds to the equivalent position usingthe amino acid numbering of a wild type TetR(B). In some examples theisolated SuR polypeptides comprise at least 10%, 20%, 30%, 40%, 50%,55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acidresidues shown in FIG. 9, wherein the amino acid residue positioncorresponds to the equivalent position using the amino acid numbering ofa wild type TetR(B). In some examples the wild type TetR(B) is SEQ IDNO: 1.

In some examples the isolated SuR polypeptide comprises a ligand bindingdomain comprising an amino acid substitution at a residue positionselected from the group consisting of position 55, 60, 64, 67, 82, 86,100, 104, 105, 108, 113, 116, 134, 135, 138, 139, 147, 151, 170, 173,174, 177 and any combination thereof, wherein the amino acid residueposition and substitution corresponds to the equivalent position usingthe amino acid numbering of a wild type TetR(B). In some examples theisolated SuR polypeptide further comprises an amino acid substitution ata residue position selected from the group consisting of 109, 112, 117,131, 137, 140, 164 and any combination thereof. In some examples thewild type TetR(B) is SEQ ID NO: 1.

In some examples the isolated SuR polypeptides comprise a ligand bindingdomain comprising at least 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% of the amino acid residues selected fromthe group consisting of:

(a) M or L at amino acid residue position 55;

(b) A, L or M at amino acid residue position 60;

(c) A, N, Q, L or H at amino acid residue position 64;

(d) M, I, L, V, F or Y at amino acid residue position 67;

(e) N, S or T at amino acid residue position 82;

(f) F, M, W or Y at amino acid residue position 86;

(g) C, V, L, M, F, W or Y at amino acid residue position 100;

(h) R, A or G at amino acid residue position 104

(i) A, I, V, F or W at amino acid residue position 105;

(j) Q or K at amino acid residue position 108;

(k) A, M, H, K, T, P or V at amino acid residue position 113;

(l) I, L, M, V, R, S, N, P or Q at amino acid residue position 116;

(m) I, L, V, M, R, S or W at amino acid residue position 134;

(n) R, S, N, Q, K or A at amino acid residue position 135;

(o) A, C, G, H, I, V, R or T at amino acid residue position 138;

(p) A, G, I, V, M, W, N, R or T at amino acid residue position 139;

(q) I, L, V, F, W, T, S or R at amino acid residue position 147;

(r) M, L, W, Y, K, R or S at amino acid residue position 151;

(s) I, L, V or A at amino acid residue position 170;

(t) A, G or V at amino acid residue position 173;

(u) L, V, W, Y, H, R, K or S at amino acid residue position 174; and,

(v) A, G, I, L, Y, K, Q or S at amino acid residue position 177,

wherein the amino acid residue position corresponds to the equivalentposition using the amino acid numbering of wild type TetR(B). In someexamples the isolated SuR polypeptides comprise at least 10%, 20%, 30%,40%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of theamino acid residues are selected from the amino acid residues listed in(a)-(v) above, wherein the amino acid residue position corresponds tothe equivalent position using the amino acid numbering of wild typeTetR(B). In some examples the wild type TetR(B) is SEQ ID NO: 1.

In some examples the isolated SuR polypeptides selected for enhancedactivity on chlorsulfuron comprise a ligand binding domain comprising atleast 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% of the amino acid residues are selected from the groupconsisting of:

(a) M at amino acid residue position 60;

(b) A or Q at amino acid residue position 64;

(c) M, F, Y, I, V or L at amino acid residue position 67;

(d) N or T at amino acid residue position 82;

(e) M at amino acid residue position 86;

(f) C or W at amino acid residue position 100;

(g) W at amino acid residue position 105;

(h) Q or K at amino acid residue position 108;

(i) M, Q, L or H at amino acid residue position 109;

(j) G, A, S or T at amino acid residue position 112;

(k) A at amino acid residue position 113;

(l) M or Q at amino acid residue position 116;

(m) M or V at amino acid residue position 134;

(n) G or R at amino acid residue position 138;

(o) N or V at amino acid residue position 139;

(p) F at amino acid residue position 147;

(q) S or L at amino acid residue position 151;

(r) A at amino acid residue position 164;

(s) A, L or V at amino acid residue position 170;

(t) A, G or V at amino acid residue position 173;

(u) L or W at amino acid residue position 174; and;

(v) K at amino acid residue position 177,

wherein the amino acid residue position corresponds to the equivalentposition using the amino acid numbering of wild type TetR(B). In someexamples the isolated SuR polypeptides comprise at least 10%, 20%, 30%,40%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of theamino acid residues are selected from the amino acid residues listed in(a)-(v) above, wherein the amino acid residue position corresponds tothe equivalent position using the amino acid numbering of wild typeTetR(B). In some examples the wild type TetR(B) is SEQ ID NO: 1.

In some examples the isolated SuR polypeptides selected for enhancedactivity on ethametsulfuron comprise a ligand binding domain comprisingat least 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% the amino acid residues are selected from the groupconsisting of:

(a) M or L at amino acid residue position 55;

(b) A at amino acid residue position 64;

(c) M, Y, F, I, L or V at amino acid residue position 67;

(d) M at amino acid residue position 86;

(e) C at amino acid residue position 100;

(f) G at amino acid residue position 104;

(g) F at amino acid residue position 105;

(h) Q or K at amino acid residue position 108;

(i) Q, M, L or H at amino acid residue position 109;

(j) S, T, G or A at amino acid residue position 112;

(k) A at amino acid residue position 113;

(l) S at amino acid residue position 116;

(m) M or L at amino acid residue position 117;

(n) M or L at amino acid residue position 131;

(o) M at amino acid residue position 134;

(p) Q at amino acid residue position 135;

(q) A or V at amino acid residue position 137;

(r) C or G at amino acid residue position 138;

(s) I at amino acid residue position 139;

(t) F or Y at amino acid residue position 140;

(u) L at amino acid residue position 147;

(v) L at amino acid residue position 151;

(w) A at amino acid residue position 164;

(x) V, A or L at amino acid residue position 170;

(y) G, A or V at amino acid residue position 173

(z) L at amino acid residue position 174; and,

(aa) N or K at amino acid residue position 177,

wherein the amino acid residue position corresponds to the equivalentposition using the amino acid numbering of wild type TetR(B). In someexamples the isolated SuR polypeptides comprise at least 10%, 20%, 30%,40%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% the aminoacid residues are selected from the amino acid residues listed in(a)-(aa) above, wherein the amino acid residue position corresponds tothe equivalent position using the amino acid numbering of wild typeTetR(B). In some examples the wild type TetR(B) is SEQ ID NO: 1.

In some examples the isolated SuR polypeptide has at least about 50%60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the ligandbinding domain of a wild type TetR(B) exemplified by amino acid residues53-207 of SEQ ID NO: 1, wherein the sequence identity is determined overthe full length of the ligand binding domain using a global alignmentmethod. In some examples the global alignment method uses the GAPalgorithm with default parameters for an amino acid sequence % identityand % similarity using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix.

In some examples the isolated SuR polypeptide has at least about 50%60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a wildtype TetR(B) exemplified by SEQ ID NO: 1, wherein the sequence identityis determined over the full length of the polypeptide using a globalalignment method. In some examples the global alignment method uses theGAP algorithm with default parameters for an amino acid sequence %identity and % similarity using GAP Weight of 8 and Length Weight of 2,and the BLOSUM62 scoring matrix.

Compositions include isolated SuR polypeptides having at least about 50%60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the ligandbinding domain of an SuR polypeptide selected from the group consistingof SEQ ID NO: 3-401, 1206-1213, 1228-1233, or 1240-1243, wherein thesequence identity is determined over the full length of the ligandbinding domain using a global alignment method. In some examples theglobal alignment method uses the GAP algorithm with default parametersfor an amino acid sequence % identity and % similarity using GAP Weightof 8 and Length Weight of 2, and the BLOSUM62 scoring matrix.

In some examples the isolated SuR polypeptide have at least about 50%60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an SuRpolypeptide selected from the group consisting of SEQ ID NO: 3-401,1206-1213, 1228-1233, or 1240-1243, wherein the sequence identity isdetermined over the full length of the polypeptide using a globalalignment method. In some examples the global alignment method uses theGAP algorithm with default parameters for an amino acid sequence %identity and % similarity using GAP Weight of 8 and Length Weight of 2,and the BLOSUM62 scoring matrix.

In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L7-1A04(SEQ ID NO:220) to generate a BLAST bit score of at least 200, 250, 275,300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625,650, 675, 700, or 750, wherein the BLAST alignment used the BLOSUM62matrix, a gap existence penalty of 11, and a gap extension penalty of 1.In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L7-1A04(SEQ ID NO:220) to generate a BLAST bit score of at least 374, whereinthe BLAST alignment used the BLOSUM62 matrix, a gap existence penalty of11, and a gap extension penalty of 1. In some examples the SuRpolypeptides comprise an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L7-1A04 (SEQ ID NO:220) togenerate a percent sequence identity of at least 50% 60%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% sequence identity, wherein the sequence identity isdetermined by BLAST alignment using the BLOSUM62 matrix, a gap existencepenalty of 11, and a gap extension penalty of 1. In some examples theSuR polypeptides comprise an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L7-1A04 (SEQ ID NO:220) togenerate a percent sequence identity of at least 88% sequence identity,wherein the sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the percent identity is determined usinga global alignment method using the GAP algorithm with defaultparameters for an amino acid sequence % identity and % similarity usingGAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix.In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L7-1A04(SEQ ID NO:220) to generate a BLAST similarity score of at least 400,425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 750, 800,850, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020,1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140,1150, 1160, 1170, 1180, 1190, or 1200 wherein the BLAST alignment usedthe BLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the SuR polypeptides comprise an aminoacid sequence that can be optimally aligned with a polypeptide sequenceof L7-1A04 (SEQ ID NO:220) to generate a BLAST e-value score of at leaste-60, e-70, e-75, e-80, e-85, e-90, e-95, e-100, e-105, e-106, e-107,e-108, e-109, e-110, e-111, e-112, e-113, e-114, e-115, e-116, e-117,e-118, e-119, e-120, or e-125, wherein the BLAST alignment used theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the polypeptide is selected from thegroup consisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or1240-1243.

In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L1-22 (SEQID NO: 7) to generate a BLAST bit score of at least 200, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650,675, 700, or 750, wherein the BLAST alignment used the BLOSUM62 matrix,a gap existence penalty of 11, and a gap extension penalty of 1. In someexamples the SuR polypeptides comprise an amino acid sequence that canbe optimally aligned with a polypeptide sequence of L1-22 (SEQ ID NO: 7)to generate a BLAST bit score of at least 387, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the SuR polypeptidescomprise an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-22 (SEQ ID NO: 7) to generate a percentsequence identity of at least 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity, wherein the sequence identity is determined byBLAST alignment using the BLOSUM62 matrix, a gap existence penalty of11, and a gap extension penalty of 1. In some examples the SuRpolypeptides comprise an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L1-22 (SEQ ID NO: 7) to generatea percent sequence identity of at least 92% sequence identity, whereinthe sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the percent identity is determined usinga global alignment method using the GAP algorithm with defaultparameters for an amino acid sequence % identity and % similarity usingGAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix.In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L1-22 (SEQID NO: 7) to generate a BLAST similarity score of at least 400, 425,450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850,900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020,1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140,1150, 1160, 1170, 1180, 1190, or 1200 wherein the BLAST alignment usedthe BLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the SuR polypeptides comprise an aminoacid sequence that can be optimally aligned with a polypeptide sequenceof L1-22 (SEQ ID NO: 7) to generate a BLAST e-value score of at leaste-60, e-70, e-75, e-80, e-85, e-90, e-95, e-100, e-105, e-106, e-107,e-108, e-109, e-110, e-111, e-112, e-113, e-114, e-115, e-116, e-117,e-118, e-119, e-120, or e-125, wherein the BLAST alignment used theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the polypeptide is selected from thegroup consisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or1240-1243.

In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L1-29 (SEQID NO: 10) to generate a BLAST bit score of at least 200, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650,675, 700, or 750, wherein the BLAST alignment used the BLOSUM62 matrix,a gap existence penalty of 11, and a gap extension penalty of 1. In someexamples the SuR polypeptides comprise an amino acid sequence that canbe optimally aligned with a polypeptide sequence of L1-29 (SEQ ID NO:10) to generate a BLAST bit score of at least 393, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the SuR polypeptidescomprise an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-29 (SEQ ID NO: 10) to generate a percentsequence identity of at least 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity, wherein the sequence identity is determined byBLAST alignment using the BLOSUM62 matrix, a gap existence penalty of11, and a gap extension penalty of 1. In some examples the percentidentity is determined using a global alignment method using the GAPalgorithm with default parameters for an amino acid sequence % identityand % similarity using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix. In some examples the SuR polypeptides comprisean amino acid sequence that can be optimally aligned with a polypeptidesequence of L1-29 (SEQ ID NO: 10) to generate a BLAST similarity scoreof at least 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675,700, 750, 800, 850, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990,1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110,1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, or 1200 wherein theBLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 11,and a gap extension penalty of 1. In some examples the SuR polypeptidescomprise an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-29 (SEQ ID NO: 10) to generate a BLASTsimilarity score of at least 1006, wherein the BLAST alignment used theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the SuR polypeptides comprise an aminoacid sequence that can be optimally aligned with a polypeptide sequenceof L1-29 (SEQ ID NO: 10) to generate a BLAST e-value score of at leaste-60, e-70, e-75, e-80, e-85, e-90, e-95, e-100, e-105, e-106, e-107,e-108, e-109, e-110, e-111, e-112, e-113, e-114, e-115, e-116, e-117,e-118, e-119, e-120, or e-125, wherein the BLAST alignment used theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the polypeptide is selected from thegroup consisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or1240-1243.

In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L1-02 (SEQID NO: 3) to generate a BLAST bit score of at least 200, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650,675, 700, or 750, wherein the BLAST alignment used the BLOSUM62 matrix,a gap existence penalty of 11, and a gap extension penalty of 1. In someexamples the SuR polypeptides comprise an amino acid sequence that canbe optimally aligned with a polypeptide sequence of L1-02 (SEQ ID NO: 3)to generate a percent sequence identity of at least 50% 60%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% sequence identity, wherein the sequenceidentity is determined by BLAST alignment using the BLOSUM62 matrix, agap existence penalty of 11, and a gap extension penalty of 1. In someexamples the percent identity is determined using a global alignmentmethod using the GAP algorithm with default parameters for an amino acidsequence % identity and % similarity using GAP Weight of 8 and LengthWeight of 2, and the BLOSUM62 scoring matrix. In some examples the SuRpolypeptides comprise an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L1-02 (SEQ ID NO: 3) to generatea BLAST similarity score of at least 400, 425, 450, 475, 500, 525, 550,575, 600, 625, 650, 675, 700, 750, 800, 850, 900, 910, 920, 930, 940,950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070,1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190,or 1200 wherein the BLAST alignment used the BLOSUM62 matrix, a gapexistence penalty of 11, and a gap extension penalty of 1. In someexamples the SuR polypeptides comprise an amino acid sequence that canbe optimally aligned with a polypeptide sequence of L1-02 (SEQ ID NO: 3)to generate a BLAST e-value score of at least e-60, e-70, e-75, e-80,e-85, e-90, e-95, e-100, e-105, e-106, e-107, e-108, e-109, e-110,e-111, e-112, e-113, e-114, e-115, e-116, e-117, e-118, e-119, e-120, ore-125, wherein the BLAST alignment used the BLOSUM62 matrix, a gapexistence penalty of 11, and a gap extension penalty of 1. In someexamples the SuR polypeptides comprise an amino acid sequence that canbe optimally aligned with a polypeptide sequence of L1-02 (SEQ ID NO: 3)to generate a BLAST e-value score of at least e-112, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the polypeptide is selectedfrom the group consisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or1240-1243.

In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L1-07 (SEQID NO: 4) to generate a BLAST bit score of at least 200, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650,675, 700, or 750, wherein the BLAST alignment used the BLOSUM62 matrix,a gap existence penalty of 11, and a gap extension penalty of 1. In someexamples the SuR polypeptides comprise an amino acid sequence that canbe optimally aligned with a polypeptide sequence of L1-07 (SEQ ID NO: 4)to generate a BLAST bit score of at least 388, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the SuR polypeptidescomprise an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-07 (SEQ ID NO: 4) to generate a percentsequence identity of at least 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity, wherein the sequence identity is determined byBLAST alignment using the BLOSUM62 matrix, a gap existence penalty of11, and a gap extension penalty of 1. n some examples the SuRpolypeptides comprise an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L1-07 (SEQ ID NO: 4) to generatea percent sequence identity of at least 93% sequence identity, whereinthe sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the percent identity is determined usinga global alignment method using the GAP algorithm with defaultparameters for an amino acid sequence % identity and % similarity usingGAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix.In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L1-07 (SEQID NO: 4) to generate a BLAST similarity score of at least 400, 425,450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850,900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020,1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140,1150, 1160, 1170, 1180, 1190, or 1200 wherein the BLAST alignment usedthe BLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the SuR polypeptides comprise an aminoacid sequence that can be optimally aligned with a polypeptide sequenceof L1-07 (SEQ ID NO: 4) to generate a BLAST similarity score of at least996, wherein the BLAST alignment used the BLOSUM62 matrix, a gapexistence penalty of 11, and a gap extension penalty of 1. In someexamples the SuR polypeptides comprise an amino acid sequence that canbe optimally aligned with a polypeptide sequence of L1-07 (SEQ ID NO: 4)to generate a BLAST e-value score of at least e-60, e-70, e-75, e-80,e-85, e-90, e-95, e-100, e-105, e-106, e-107, e-108, e-109, e-110,e-111, e-112, e-113, e-114, e-115, e-116, e-117, e-118, e-119, e-120, ore-125, wherein the BLAST alignment used the BLOSUM62 matrix, a gapexistence penalty of 11, and a gap extension penalty of 1. In someexamples the SuR polypeptides comprise an amino acid sequence that canbe optimally aligned with a polypeptide sequence of L1-07 (SEQ ID NO: 4)to generate a BLAST e-value score of at least e-111, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the polypeptide is selectedfrom the group consisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or1240-1243.

In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L1-20 (SEQID NO: 6) to generate a BLAST bit score of at least 200, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650,675, 700, or 750, wherein the BLAST alignment used the BLOSUM62 matrix,a gap existence penalty of 11, and a gap extension penalty of 1. In someexamples the SuR polypeptides comprise an amino acid sequence that canbe optimally aligned with a polypeptide sequence of L1-20 (SEQ ID NO: 6)to generate a percent sequence identity of at least 50% 60%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% sequence identity, wherein the sequenceidentity is determined by BLAST alignment using the BLOSUM62 matrix, agap existence penalty of 11, and a gap extension penalty of 1. In someexamples the SuR polypeptides comprise an amino acid sequence that canbe optimally aligned with a polypeptide sequence of L1-20 (SEQ ID NO: 6)to generate a percent sequence identity of at least 93% sequenceidentity, wherein the sequence identity is determined by BLAST alignmentusing the BLOSUM62 matrix, a gap existence penalty of 11, and a gapextension penalty of 1. In some examples the percent identity isdetermined using a global alignment method using the GAP algorithm withdefault parameters for an amino acid sequence % identity and %similarity using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix. In some examples the SuR polypeptides comprisean amino acid sequence that can be optimally aligned with a polypeptidesequence of L1-20 (SEQ ID NO: 6) to generate a BLAST similarity score ofat least 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675,700, 750, 800, 850, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990,1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110,1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, or 1200 wherein theBLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 11,and a gap extension penalty of 1. In some examples the SuR polypeptidescomprise an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-20 (SEQ ID NO: 6) to generate a BLAST e-valuescore of at least e-60, e-70, e-75, e-80, e-85, e-90, e-95, e-100,e-105, e-106, e-107, e-108, e-109, e-110, e-111, e-112, e-113, e-114,e-115, e-116, e-117, e-118, e-119, e-120, or e-125, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the SuR polypeptidescomprise an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-20 (SEQ ID NO: 6) to generate a BLAST e-valuescore of at least e-111, wherein the BLAST alignment used the BLOSUM62matrix, a gap existence penalty of 11, and a gap extension penalty of 1.In some examples the polypeptide is selected from the group consistingof SEQ ID NO: 3-401, 1206-1213, 1228-1233, or 1240-1243.

In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L1-44 (SEQID NO: 13) to generate a BLAST bit score of at least 200, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650,675, 700, or 750, wherein the BLAST alignment used the BLOSUM62 matrix,a gap existence penalty of 11, and a gap extension penalty of 1. In someexamples the SuR polypeptides comprise an amino acid sequence that canbe optimally aligned with a polypeptide sequence of L1-44 (SEQ ID NO:13) to generate a percent sequence identity of at least 50% 60%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity, wherein the sequenceidentity is determined by BLAST alignment using the BLOSUM62 matrix, agap existence penalty of 11, and a gap extension penalty of 1. In someexamples the SuR polypeptides comprise an amino acid sequence that canbe optimally aligned with a polypeptide sequence of L1-44 (SEQ ID NO:13) to generate a percent sequence identity of at least 93% sequenceidentity, wherein the sequence identity is determined by BLAST alignmentusing the BLOSUM62 matrix, a gap existence penalty of 11, and a gapextension penalty of 1. In some examples the percent identity isdetermined using a global alignment method using the GAP algorithm withdefault parameters for an amino acid sequence % identity and %similarity using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix. In some examples the SuR polypeptides comprisean amino acid sequence that can be optimally aligned with a polypeptidesequence of L1-44 (SEQ ID NO: 13) to generate a BLAST similarity scoreof at least 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675,700, 750, 800, 850, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990,1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110,1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, or 1200 wherein theBLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 11,and a gap extension penalty of 1. In some examples the SuR polypeptidescomprise an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-44 (SEQ ID NO: 13) to generate a BLASTe-value score of at least e-60, e-70, e-75, e-80, e-85, e-90, e-95,e-100, e-105, e-106, e-107, e-108, e-109, e-110, e-111, e-112, e-113,e-114, e-115, e-116, e-117, e-118, e-119, e-120, or e-125, wherein theBLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 11,and a gap extension penalty of 1. In some examples the polypeptide isselected from the group consisting of SEQ ID NO: 3-401, 1206-1213,1228-1233, or 1240-1243.

In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L6-3A09(SEQ ID NO: 1228) to generate a BLAST bit score of at least 200, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600,625, 650, 675, 700, or 750, wherein the BLAST alignment used theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the SuR polypeptides comprise an aminoacid sequence that can be optimally aligned with a polypeptide sequenceof L6-3A09 (SEQ ID NO: 1228) to generate a BLAST bit score of at least381, wherein the BLAST alignment used the BLOSUM62 matrix, a gapexistence penalty of 11, and a gap extension penalty of 1. In someexamples the SuR polypeptides comprise an amino acid sequence that canbe optimally aligned with a polypeptide sequence of L6-3A09 (SEQ ID NO:1228) to generate a percent sequence identity of at least 50% 60%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity, wherein the sequenceidentity is determined by BLAST alignment using the BLOSUM62 matrix, agap existence penalty of 11, and a gap extension penalty of 1. In someexamples the percent identity is determined using a global alignmentmethod using the GAP algorithm with default parameters for an amino acidsequence % identity and % similarity using GAP Weight of 8 and LengthWeight of 2, and the BLOSUM62 scoring matrix. In some examples the SuRpolypeptides comprise an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L6-3A09 (SEQ ID NO: 1228) togenerate a BLAST similarity score of at least 400, 425, 450, 475, 500,525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850, 900, 910, 920,930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050,1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170,1180, 1190, or 1200 wherein the BLAST alignment used the BLOSUM62matrix, a gap existence penalty of 11, and a gap extension penalty of 1.In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L6-3A09(SEQ ID NO: 1228) to generate a BLAST similarity score of at least 978wherein the BLAST alignment used the BLOSUM62 matrix, a gap existencepenalty of 11, and a gap extension penalty of 1. In some examples theSuR polypeptides comprise an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L6-3A09 (SEQ ID NO: 1228) togenerate a BLAST e-value score of at least e-60, e-70, e-75, e-80, e-85,e-90, e-95, e-100, e-105, e-106, e-107, e-108, e-109, e-110, e-111,e-112, e-113, e-114, e-115, e-116, e-117, e-118, e-119, e-120, or e-125,wherein the BLAST alignment used the BLOSUM62 matrix, a gap existencepenalty of 11, and a gap extension penalty of 1. In some examples theSuR polypeptides comprise an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L6-3A09 (SEQ ID NO: 1228) togenerate a BLAST e-value score of at least e-108, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the polypeptide is selectedfrom the group consisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or1240-1243.

In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L6-3H02(SEQ ID NO: 94) to generate a BLAST bit score of at least 200, 250, 275,300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625,650, 675, 700, or 750, wherein the BLAST alignment used the BLOSUM62matrix, a gap existence penalty of 11, and a gap extension penalty of 1.In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L6-3H02(SEQ ID NO: 94) to generate a percent sequence identity of at least 50%60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, wherein thesequence identity is determined by BLAST alignment using the BLOSUM62matrix, a gap existence penalty of 11, and a gap extension penalty of 1.In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L6-3H02(SEQ ID NO: 94) to generate a percent sequence identity of at least 90%sequence identity, wherein the sequence identity is determined by BLASTalignment using the BLOSUM62 matrix, a gap existence penalty of 11, anda gap extension penalty of 1. In some examples the percent identity isdetermined using a global alignment method using the GAP algorithm withdefault parameters for an amino acid sequence % identity and %similarity using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix. In some examples the SuR polypeptides comprisean amino acid sequence that can be optimally aligned with a polypeptidesequence of L6-3H02 (SEQ ID NO: 94) to generate a BLAST similarity scoreof at least 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675,700, 750, 800, 850, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990,1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110,1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, or 1200 wherein theBLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 11,and a gap extension penalty of 1. In some examples the SuR polypeptidescomprise an amino acid sequence that can be optimally aligned with apolypeptide sequence of L6-3H02 (SEQ ID NO: 94) to generate a BLASTe-value score of at least e-60, e-70, e-75, e-80, e-85, e-90, e-95,e-100, e-105, e-106, e-107, e-108, e-109, e-110, e-111, e-112, e-113,e-114, e-115, e-116, e-117, e-118, e-119, e-120, or e-125, wherein theBLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 11,and a gap extension penalty of 1. In some examples the polypeptide isselected from the group consisting of SEQ ID NO: 3-401, 1206-1213,1228-1233, or 1240-1243.

In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L7-4E03(SEQ ID NO: 1229) to generate a BLAST bit score of at least 200, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600,625, 650, 675, 700, or 750, wherein the BLAST alignment used theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the SuR polypeptides comprise an aminoacid sequence that can be optimally aligned with a polypeptide sequenceof L7-4E03 (SEQ ID NO: 1229) to generate a BLAST bit score of at least368, wherein the BLAST alignment used the BLOSUM62 matrix, a gapexistence penalty of 11, and a gap extension penalty of 1. In someexamples the SuR polypeptides comprise an amino acid sequence that canbe optimally aligned with a polypeptide sequence of L7-4E03 (SEQ ID NO:1229) to generate a percent sequence identity of at least 50% 60%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity, wherein the sequenceidentity is determined by BLAST alignment using the BLOSUM62 matrix, agap existence penalty of 11, and a gap extension penalty of 1. In someexamples the percent identity is determined using a global alignmentmethod using the GAP algorithm with default parameters for an amino acidsequence % identity and % similarity using GAP Weight of 8 and LengthWeight of 2, and the BLOSUM62 scoring matrix. In some examples the SuRpolypeptides comprise an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L7-4E03 (SEQ ID NO: 1229) togenerate a BLAST similarity score of at least 400, 425, 450, 475, 500,525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850, 900, 910, 920,930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050,1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170,1180, 1190, or 1200 wherein the BLAST alignment used the BLOSUM62matrix, a gap existence penalty of 11, and a gap extension penalty of 1.In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L7-4E03(SEQ ID NO: 1229) to generate a BLAST similarity score of at least 945wherein the BLAST alignment used the BLOSUM62 matrix, a gap existencepenalty of 11, and a gap extension penalty of 1. In some examples theSuR polypeptides comprise an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L7-4E03 (SEQ ID NO: 1229) togenerate a BLAST e-value score of at least e-60, e-70, e-75, e-80, e-85,e-90, e-95, e-100, e-105, e-106, e-107, e-108, e-109, e-110, e-111,e-112, e-113, e-114, e-115, e-116, e-117, e-118, e-119, e-120, or e-125,wherein the BLAST alignment used the BLOSUM62 matrix, a gap existencepenalty of 11, and a gap extension penalty of 1. In some examples theSuR polypeptides comprise an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L7-4E03 (SEQ ID NO: 1229) togenerate a BLAST e-value score of at least e-105, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the polypeptide is selectedfrom the group consisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or1240-1243.

In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L10-84(B12)(SEQ ID NO: 1230) to generate a BLAST bit score of at least 200, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600,625, 650, 675, 700, or 750, wherein the BLAST alignment used theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the SuR polypeptides comprise an aminoacid sequence that can be optimally aligned with a polypeptide sequenceof L10-84(B12) (SEQ ID NO: 1230) to generate a percent sequence identityof at least 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity, wherein the sequence identity is determined by BLAST alignmentusing the BLOSUM62 matrix, a gap existence penalty of 11, and a gapextension penalty of 1. In some examples the SuR polypeptides comprisean amino acid sequence that can be optimally aligned with a polypeptidesequence of L10-84(B12) (SEQ ID NO: 1230) to generate a percent sequenceidentity of at least 86% sequence identity, wherein the sequenceidentity is determined by BLAST alignment using the BLOSUM62 matrix, agap existence penalty of 11, and a gap extension penalty of 1. In someexamples the percent identity is determined using a global alignmentmethod using the GAP algorithm with default parameters for an amino acidsequence % identity and % similarity using GAP Weight of 8 and LengthWeight of 2, and the BLOSUM62 scoring matrix. In some examples the SuRpolypeptides comprise an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L10-84(B12) (SEQ ID NO: 1230) togenerate a BLAST similarity score of at least 400, 425, 450, 475, 500,525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850, 900, 910, 920,930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050,1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170,1180, 1190, or 1200 wherein the BLAST alignment used the BLOSUM62matrix, a gap existence penalty of 11, and a gap extension penalty of 1.In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L10-84(B12)(SEQ ID NO: 1230) to generate a BLAST e-value score of at least e-60,e-70, e-75, e-80, e-85, e-90, e-95, e-100, e-105, e-106, e-107, e-108,e-109, e-110, e-111, e-112, e-113, e-114, e-115, e-116, e-117, e-118,e-119, e-120, or e-125, wherein the BLAST alignment used the BLOSUM62matrix, a gap existence penalty of 11, and a gap extension penalty of 1.In some examples the polypeptide is selected from the group consistingof SEQ ID NO: 3-401, 1206-1213, 1228-1233, or 1240-1243.

In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L13-46 (SEQID NO: 1231) to generate a BLAST bit score of at least 200, 250, 275,300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625,650, 675, 700, or 750, wherein the BLAST alignment used the BLOSUM62matrix, a gap existence penalty of 11, and a gap extension penalty of 1.In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L13-46 (SEQID NO: 1231) to generate a BLAST bit score of at least 320, wherein theBLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 11,and a gap extension penalty of 1. In some examples the SuR polypeptidescomprise an amino acid sequence that can be optimally aligned with apolypeptide sequence of L13-46 (SEQ ID NO: 1231) to generate a percentsequence identity of at least 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity, wherein the sequence identity is determined byBLAST alignment using the BLOSUM62 matrix, a gap existence penalty of11, and a gap extension penalty of 1. In some examples the SuRpolypeptides comprise an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L13-46 (SEQ ID NO: 1231) togenerate a percent sequence identity of at least 86% sequence identity,wherein the sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the percent identity is determined usinga global alignment method using the GAP algorithm with defaultparameters for an amino acid sequence % identity and % similarity usingGAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix.In some examples the SuR polypeptides comprise an amino acid sequencethat can be optimally aligned with a polypeptide sequence of L13-46 (SEQID NO: 1231) to generate a BLAST similarity score of at least 400, 425,450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850,900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020,1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140,1150, 1160, 1170, 1180, 1190, or 1200 wherein the BLAST alignment usedthe BLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the SuR polypeptides comprise an aminoacid sequence that can be optimally aligned with a polypeptide sequenceof L13-46 (SEQ ID NO: 1231) to generate a BLAST similarity score of atleast 819 wherein the BLAST alignment used the BLOSUM62 matrix, a gapexistence penalty of 11, and a gap extension penalty of 1. In someexamples the SuR polypeptides comprise an amino acid sequence that canbe optimally aligned with a polypeptide sequence of L13-46 (SEQ ID NO:1231) to generate a BLAST e-value score of at least e-60, e-70, e-75,e-80, e-85, e-90, e-95, e-100, e-105, e-106, e-107, e-108, e-109, e-110,e-111, e-112, e-113, e-114, e-115, e-116, e-117, e-118, e-119, e-120, ore-125, wherein the BLAST alignment used the BLOSUM62 matrix, a gapexistence penalty of 11, and a gap extension penalty of 1. In someexamples the SuR polypeptides comprise an amino acid sequence that canbe optimally aligned with a polypeptide sequence of L13-46 (SEQ ID NO:1231) to generate a BLAST e-value score of at least e-90, wherein theBLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 11,and a gap extension penalty of 1. In some examples the polypeptide isselected from the group consisting of SEQ ID NO: 3-401, 1206-1213,1228-1233, or 1240-1243.

In some examples the isolated SuR polypeptides comprise a ligand bindingdomain from a polypeptide selected from the group consisting of SEQ IDNO: 3-401, 1206-1213, 1228-1233, or 1240-1243. In some examples theisolated SuR polypeptides comprise an amino acid sequence selected fromthe group consisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or1240-1243. In some examples the isolated SuR polypeptide is selectedfrom the group consisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or1240-1243, and the sulfonylurea compound is selected from the groupconsisting of a chlorsulfuron, an ethametsulfuron, a metsulfuron, asulfometuron, a tribenuron, a chlorimuron, a nicosulfuron, a rimsulfuronand a thifensulfuron.

In some examples the isolated SuR polypeptides have an equilibriumbinding constant for a sulfonylurea compound greater than 0.1 nM andless than 10 μM. In some examples the isolated SuR polypeptide has anequilibrium binding constant for a sulfonylurea compound of at least 0.1nM, 0.5 nM, 1 nM, 10 nM, 50 nM, 100 nM, 250 nM, 500 nM, 750 nM, 1 μM, 5μM, 7 μM but less than 10 μM. In some examples the isolated SuRpolypeptide has an equilibrium binding constant for a sulfonylureacompound of at least 0.1 nM, 0.5 nM, 1 nM, 10 nM, 50 nM, 100 nM, 250 nM,500 nM, 750 nM but less than 1 μM. In some examples the isolated SuRpolypeptide has an equilibrium binding constant for a sulfonylureacompound greater than 0 nM, but less than 0.1 nM, 0.5 nM, 1 nM, 10 nM,50 nM, 100 nM, 250 nM, 500 nM, 750 nM, 1 μM, 5 μM, 7 μM or 10 μM. Insome examples the sulfonylurea compound is a chlorsulfuron, anethametsulfuron, a metsulfuron, a sulfometuron, a tribenuron, achlorimuron, a nicosulfuron, a rimsulfuron and/or a thifensulfuron.

In some examples the isolated SuR polypeptides have an equilibriumbinding constant for an operator sequence greater than 0.1 nM and lessthan 10 μM. In some examples the isolated SuR polypeptide has anequilibrium binding constant for an operator sequence of at least 0.1nM, 0.5 nM, 1 nM, 10 nM, 50 nM, 100 nM, 250 nM, 500 nM, 750 nM, 1 μM, 5μM, 7 μM but less than 10 μM. In some examples the isolated SuRpolypeptide has an equilibrium binding constant for an operator sequenceof at least 0.1 nM, 0.5 nM, 1 nM, 10 nM, 50 nM, 100 nM, 250 nM, 500 nM,750 nM but less than 1 μM. In some examples the isolated SuR polypeptidehas an equilibrium binding constant for an operator sequence greaterthan 0 nM, but less than 0.1 nM, 0.5 nM, 1 nM, 10 nM, 50 nM, 100 nM, 250nM, 500 nM, 750 nM, 1 μM, 5 μM, 7 μM or 10 μM. In some examples theoperator sequence is a Tet operator sequence. In some examples the Tetoperator sequence is a TetR(A) operator sequence, a TetR(B) operatorsequence, a TetR(D) operator sequence, TetR(E) operator sequence, aTetR(H) operator sequence, or a functional derivative thereof.

The isolated SuR polypeptides specifically bind to a sulfonylureacompound. Sulfonylurea molecules comprise a sulfonylurea moiety(—S(O)2NHC(O)NH(R)—). In sulfonylurea herbicides the sulfonyl end of thesulfonylurea moiety is connected either directly or by way of an oxygenatom or an optionally substituted amino or methylene group to atypically substituted cyclic or acyclic group. At the opposite end ofthe sulfonylurea bridge, the amino group, which may have a substituentsuch as methyl (R being CH3) instead of hydrogen, is connected to aheterocyclic group, typically a symmetric pyrimidine or triazine ring,having one or two substituents such as methyl, ethyl, trifluoromethyl,methoxy, ethoxy, methylamino, dimethylamino, ethylamino and thehalogens. Sulfonylurea herbicides can be in the form of the free acid ora salt. In the free acid form the sulfonamide nitrogen on the bridge isnot deprotonated (i.e., —S(O)2NHC(O)NH(R)—), while in the salt form thesulfonamide nitrogen atom on the bridge is deprotonated (i.e.,—S(O)2NC(O)NH(R)—), and a cation is present, typically of an alkalimetal or alkaline earth metal, most commonly sodium or potassium.Sulfonylurea compounds include, for example, compound classes such aspyrimidinylsulfonylurea compounds, triazinylsulfonylurea compounds,thiadiazolylurea compounds, and pharmaceuticals such as antidiabeticdrugs, as well as salts and other derivatives thereof. Examples ofpyrimidinylsulfonylurea compounds include amidosulfuron, azimsulfuron,bensulfuron, bensulfuron-methyl, chlorimuron, chlorimuron-ethyl,cyclosulfamuron, ethoxysulfuron, flazasulfuron, flucetosulfuron,flupyrsulfuron, flupyrsulfuron-methyl, foramsulfuron, halosulfuron,halosulfuron-methyl, imazosulfuron, mesosulfuron, mesosulfuron-methyl,nicosulfuron, orthosulfamuron, oxasulfuron, primisulfuron, primisulfuron-methyl, pyrazosulfuron, pyrazosulfuron-ethyl, rimsulfuron,sulfometuron, sulfometuron-methyl, sulfosulfuron, trifloxysulfuron andsalts and derivatives thereof. Examples of triazinylsulfonylureacompounds include chlorsulfuron, cinosulfuron, ethametsulfuron,ethametsulfuron-methyl, iodosulfuron, iodosulfuron-methyl, metsulfuron,metsulfuron-methyl, prosulfuron, thifensulfuron, thifensulfuron-methyl,triasulfuron, tribenuron, tribenuron-methyl, triflusulfuron,triflusulfuron-methyl, tritosulfuron and salts and derivatives thereof.Examples of thiadiazolylurea compounds include buthiuron, ethidimuron,tebuthiuron, thiazafluoron, thidiazuron and salts and derivativesthereof. Examples of antidiabetic drugs include acetohexamide,chlorpropamide, tolbutamide, tolazamide, glipizide, gliclazide,glibenclamide (glyburide), gliquidone, glimepiride and salts andderivatives thereof. In some examples the isolated SuR polypeptidesspecifically bind to more than one sulfonylurea compound. In someexamples the sulfonylurea compound is selected from the group consistingof chlorsulfuron, ethametsulfuron-methyl, metsulfuron-methyl,thifensulfuron-methyl, sulfometuron-methyl, tribenuron-methyl,chlorimuron-ethyl, nicosulfuron, and rimsulfuron.

Compositions also include isolated polynucleotides encoding SuRpolypeptides that specifically bind to a tetracycline operator, whereinthe specific binding is regulated by a sulfonylurea compound. In someexamples the isolated polynucleotides encode sulfonylurea repressor(SuR) polypeptides comprising an amino acid substitution in the ligandbinding domain of a wild type tetracycline repressor protein. In class Band D wild type TetR proteins, amino acid residues 6-52 represent theDNA binding domain. The remainder of the protein is involved in ligandbinding and subsequent allosteric modification. For class B TetRresidues 53-207 represent the ligand binding domain, while residues53-218 comprise the ligand binding domain for the class D TetR. In someexamples the isolated polynucleotides encode SuR polypeptides comprisingan amino acid substitution in the ligand binding domain of a wild typeTetR(B) protein. In some examples the polynucleotides encode SuRpolypeptides comprising an amino acid substitution in the ligand bindingdomain of a wild type TetR(B) protein of SEQ ID NO: 1.

In some examples the isolated polynucleotides encode SuR polypeptidescomprising an amino acid, or any combination of amino acids, selectedfrom the amino acid diversity shown in FIG. 9, wherein the amino acidresidue position corresponds to the equivalent position using the aminoacid numbering of a wild type TetR(B) exemplified by SEQ ID NO: 1. Insome examples the isolated polynucleotides encode SuR polypeptidescomprising a ligand binding domain comprising at least 10%, 20%, 30%,40%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of theamino acid residues shown in FIG. 9, wherein the amino acid residueposition corresponds to the equivalent position using the amino acidnumbering of wild type TetR(B). In some examples the isolatedpolynucleotides encode SuR polypeptides comprising at least 10%, 20%,30%, 40%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% ofthe amino acid residues shown in FIG. 9, wherein the amino acid residueposition corresponds to the equivalent position using the amino acidnumbering of wild type TetR(B). In some examples the wild type TetR(B)is SEQ ID NO: 1.

In some examples the isolated polynucleotides encode SuR polypeptidescomprising a ligand binding domain comprising an amino acid substitutionat a residue position selected from the group consisting of position 55,60, 64, 67, 82, 86, 100, 104, 105, 108, 113, 116, 134, 135, 138, 139,147, 151, 170, 173, 174, 177 and any combination thereof, wherein theamino acid residue position and substitution corresponds to theequivalent position using the amino acid numbering of a wild typeTetR(B). In some examples the isolated polynucleotides encode SuRpolypeptides further comprising an amino acid substitution at a residueposition selected from the group consisting of 109, 112, 117, 131, 137,140, 164 and any combination thereof. In some examples the wild typeTetR(B) polypeptide sequence is SEQ ID NO: 1.

In some examples the isolated polynucleotides encode SuR polypeptideshaving a ligand binding domain comprising at least 10%, 20%, 30%, 40%,50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the aminoacid residues are selected from the group consisting of:

-   (a) M or L at amino acid residue position 55;-   (b) A, L or M at amino acid residue position 60;-   (c) A, N, Q, L or H at amino acid residue position 64;-   (d) M, I, L, V, F or Y at amino acid residue position 67;-   (e) N, S or T at amino acid residue position 82;-   (f) F, M, W or Y at amino acid residue position 86;-   (g) C, V, L, M, F, W or Y at amino acid residue position 100;-   (h) R, A or G at amino acid residue position 104-   (i) A, I, V, F or W at amino acid residue position 105;-   (j) Q or K at amino acid residue position 108;-   (k) A, M, H, K, T, P or V at amino acid residue position 113;-   (l) I, L, M, V, R, S, N, P or Q at amino acid residue position 116;-   (m) I, L, V, M, R, S or W at amino acid residue position 134;-   (n) R, S, N, Q, K or A at amino acid residue position 135;-   (o) A, C, G, H, I, V, R or T at amino acid residue position 138;-   (p) A, G, I, V, M, W, N, R or T at amino acid residue position 139;-   (q) I, L, V, F, W, T, S or R at amino acid residue position 147;-   (r) M, L, W, Y, K, R or S at amino acid residue position 151;-   (s) I, L, V or A at amino acid residue position 170;-   (t) A, G or V at amino acid residue position 173;-   (u) L, V, W, Y, H, R, K or S at amino acid residue position 174;    and,-   (v) A, G, I, L, Y, K, Q or S at amino acid residue position 177,    wherein the amino acid residue position corresponds to the    equivalent position using the amino acid numbering of wild type    TetR(B). In some examples the isolated SuR polynucleotides encode    SuR polypeptides comprising at least 10%, 20%, 30%, 40%, 50%, 55%,    60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,    77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,    90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the    amino acid residues are selected from the amino acid residues listed    in (a)-(v) above, wherein the amino acid residue position    corresponds to the equivalent position using the amino acid    numbering of wild type TetR(B). In some examples the wild type    TetR(B) is SEQ ID NO: 1.

In some examples the isolated polynucleotides encode SuR polypeptidesselected for enhanced activity on chlorsulfuron having a ligand bindingdomain comprising at least 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% of the amino acid residues are selectedfrom the group consisting of:

-   (a) M at amino acid residue position 60;-   (b) A or Q at amino acid residue position 64;-   (c) M, F, Y, I, V or L at amino acid residue position 67;-   (d) N or T at amino acid residue position 82;-   (e) M at amino acid residue position 86;-   (f) C or W at amino acid residue position 100;-   (g) W at amino acid residue position 105;-   (h) Q or K at amino acid residue position 108;-   (i) M, Q, L or H at amino acid residue position 109;-   (j) G, A, S or T at amino acid residue position 112;-   (k) A at amino acid residue position 113;-   (l) M or Q at amino acid residue position 116;-   (m) M or V at amino acid residue position 134;-   (n) G or R at amino acid residue position 138;-   (o) N or V at amino acid residue position 139;-   (p) F at amino acid residue position 147;-   (q) S or L at amino acid residue position 151;-   (r) A at amino acid residue position 164;-   (s) A, L or V at amino acid residue position 170;-   (t) A, G or V at amino acid residue position 173;-   (u) L or W at amino acid residue position 174; and;-   (v) K at amino acid residue position 177,    wherein the amino acid residue position corresponds to the    equivalent position using the amino acid numbering of wild type    TetR(B). In some examples the isolated SuR polynucleotides encode    SuR polypeptides comprising at least 10%, 20%, 30%, 40%, 50%, 55%,    60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,    77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,    90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the    amino acid residues are selected from the amino acid residues listed    in (a)-(v) above wherein the amino acid residue position corresponds    to the equivalent position using the amino acid numbering of wild    type TetR(B). In some examples the wild type TetR(B) is SEQ ID NO:    1.

In some examples the isolated polynucleotides encode SuR polypeptidesselected for enhanced activity on ethametsulfuron having a ligandbinding domain comprising at least 10%, 20%, 30%, 40%, 50%, 55%, 60%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid residues areselected from the group consisting of:

-   (a) M or L at amino acid residue position 55;-   (b) A at amino acid residue position 64;-   (c) M, Y, F, I, L or V at amino acid residue position 67;-   (d) M at amino acid residue position 86;-   (e) C at amino acid residue position 100;-   (f) G at amino acid residue position 104;-   (g) F at amino acid residue position 105;-   (h) Q or K at amino acid residue position 108;-   (i) Q, M, L or H at amino acid residue position 109;-   (j) S, T, G or A at amino acid residue position 112;-   (k) A at amino acid residue position 113;-   (l) S at amino acid residue position 116;-   (m) M or L at amino acid residue position 117;-   (n) M or L at amino acid residue position 131;-   (o) M at amino acid residue position 134;-   (p) Q at amino acid residue position 135;-   (q) A or V at amino acid residue position 137;-   (r) C or G amino acid residue 138;-   (s) I at amino acid residue position 139;-   (t) F or Y at amino acid residue position 140;-   (u) L at amino acid residue position 147;-   (v) L at amino acid residue position 151;-   (w) A at amino acid residue position 164;-   (x) V, A or L at amino acid residue position 170;-   (y) G, A or V at amino acid residue position 173;-   (z) L at amino acid residue position 174; and,-   (aa) N or K at amino acid residue position 177,    wherein the amino acid residue position corresponds to the    equivalent position using the amino acid numbering of wild type    TetR(B). In some examples the isolated SuR polynucleotides encode    SuR polypeptides comprising at least 10%, 20%, 30%, 40%, 50%, 55%,    60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,    77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,    90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the    amino acid residues are selected from the amino acid residues listed    in (a)-(aa) above, wherein the amino acid residue position    corresponds to the equivalent position using the amino acid    numbering of wild type TetR(B). In some examples the wild type    TetR(B) is SEQ ID NO: 1.

In some examples the isolated polynucleotides encode SuR polypeptideshaving at least about 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to the ligand binding domain shown as amino acidresidues 53-207 of SEQ ID NO: 1, wherein the sequence identity isdetermined over the full length of the ligand binding domain using aglobal alignment method. In some examples the global alignment method isGAP, wherein the default parameters are for an amino acid sequence %identity and % similarity using GAP Weight of 8 and Length Weight of 2,and the BLOSUM62 scoring matrix.

In some examples the isolated polynucleotides encode SuR polypeptideshaving at least about 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to SEQ ID NO: 1, wherein the sequence identity isdetermined over the full length of the polypeptide using a globalalignment method. In some examples the global alignment method is GAP,wherein the default parameters are for an amino acid sequence % identityand % similarity using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix.

In some examples the isolated polynucleotides include nucleic acidsequences that selectively hybridize under stringent hybridizationconditions to a polynucleotide encoding a SuR polypeptide.Polynucleotides that selectively hybridize are polynucleotides whichbind to a target sequence at a level of at least 2-fold over backgroundas compared to hybridization to a non-target sequence. Stringentconditions are sequence-dependent and condition-dependent. Typicalstringent conditions are those in which the salt concentration about0.01 to 1.0 M at pH 7.0-8.3 at 30° C. for short probes (e.g., 10 to 50nucleotides) or about 60° C. for long probes (e.g., greater than 50nucleotides). Stringent conditions may include formamide or otherdestabilizing agents. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is impacted by post-hybridization wash conditions, typicallyvia ionic strength and temperature. For DNA-DNA hybrids, the T_(m) canbe approximated from the equation of Meinkoth and Wahl, (1984) Anal.Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. An extensive guide to thehybridization of nucleic acids is found in Tijssen, LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes, Part I, Chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays”, Elsevier,New York (1993); and Current Protocols in Molecular Biology, Chapter 2,Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, NewYork (1995). In some examples, the isolated polynucleotides encoding SuRpolypeptides specifically hybridize to a polynucleotide of SEQ ID NO:434-832, 1214-1221, 1234-1239, or 1244-1247 under moderately stringentconditions or under highly stringent conditions.

In some examples the isolated polynucleotides encode an SuR polypeptidecomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L7-1A04 (SEQ ID NO: 220) to generate a BLAST bitscore of at least 200, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, or 750, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the isolatedpolynucleotides encode an SuR polypeptide comprising an amino acidsequence that can be optimally aligned with a polypeptide sequence ofL7-1A04 (SEQ ID NO: 220) to generate a BLAST bit score of at least 374,wherein the BLAST alignment used the BLOSUM62 matrix, a gap existencepenalty of 11, and a gap extension penalty of 1. In some examples theisolated polynucleotides encode SuR polypeptides comprising an aminoacid sequence that can be optimally aligned with a polypeptide sequenceof L7-1A04 (SEQ ID NO:220) to generate a percent sequence identity of atleast 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity,wherein the sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L7-1A04 (SEQ ID NO:220) togenerate a percent sequence identity of at least 88% sequence identity,wherein the sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the percent identity is determined usinga global alignment method using the GAP algorithm with defaultparameters for an amino acid sequence % identity and % similarity usingGAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix.In some examples the isolated polynucleotides encode SuR polypeptidescomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L7-1A04 (SEQ ID NO:220) to generate a BLASTsimilarity score of at least 400, 425, 450, 475, 500, 525, 550, 575,600, 625, 650, 675, 600, 750, 800, 850, 900, 910, 920, 930, 940, 950,960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070,1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190,or 1200, wherein BLAST alignment used the BLOSUM62 matrix, a gapexistence penalty of 11, and a gap extension penalty of 1. In someexamples the isolated polynucleotides encode SuR polypeptides comprisingan amino acid sequence that can be optimally aligned with a polypeptidesequence of L7-1A04 (SEQ ID NO:220) to generate a BLAST e-value score ofat least e-60, e-70, e-80, e-85, e-90, e-95, e-100, e-105, e-106, e-107,e-108, e-109, e-110, e-111, e-112, e-113, e-114, e-115, e-116, e-117,e-118, e-119, e-120, or e-125, wherein BLAST alignment used the BLOSUM62matrix, a gap existence penalty of 11, and a gap extension penalty of 1.In some examples isolated polynucleotide encodes a polypeptide selectedfrom the group consisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or1240-1243. In some examples the isolated polynucleotides comprise apolynucleotide sequence of SEQ ID NO: 434-832, 1214-1221, 1234-1239, or1244-1247, or the complementary polynucleotide thereof.

In some examples the isolated polynucleotides encode an SuR polypeptidecomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-22 (SEQ ID NO: 7) to generate a BLAST bitscore of at least 200, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, or 750, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the isolatedpolynucleotides encode an SuR polypeptide comprising an amino acidsequence that can be optimally aligned with a polypeptide sequence ofL1-22 (SEQ ID NO: 7) to generate a BLAST bit score of at least 387,wherein the BLAST alignment used the BLOSUM62 matrix, a gap existencepenalty of 11, and a gap extension penalty of 1. In some examples theisolated polynucleotides encode SuR polypeptides comprising an aminoacid sequence that can be optimally aligned with a polypeptide sequenceof L1-22 (SEQ ID NO: 7) to generate a percent sequence identity of atleast 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity,wherein the sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L1-22 (SEQ ID NO: 7) to generatea percent sequence identity of at least 92% sequence identity, whereinthe sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the percent sequence identity isdetermined using a global alignment method using the GAP algorithm withdefault parameters for an amino acid sequence % identity and %similarity using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix. In some examples the isolated polynucleotidesencode SuR polypeptides comprising an amino acid sequence that can beoptimally aligned with a polypeptide sequence of L1-22 (SEQ ID NO: 7) togenerate a BLAST similarity score of at least 400, 425, 450, 475, 500,525, 550, 575, 600, 625, 650, 675, 600, 750, 800, 850, 900, 910, 920,930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050,1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170,1180, 1190, or 1200, wherein BLAST alignment used the BLOSUM62 matrix, agap existence penalty of 11, and a gap extension penalty of 1. In someexamples the isolated polynucleotides encode SuR polypeptides comprisingan amino acid sequence that can be optimally aligned with a polypeptidesequence of L1-22 (SEQ ID NO: 7) to generate a BLAST e-value score of atleast e-60, e-70, e-80, e-85, e-90, e-95, e-100, e-105, e-106, e-107,e-108, e-109, e-110, e-111, e-112, e-113, e-114, e-115, e-116, e-117,e-118, e-119, e-120, or e-125, wherein BLAST alignment used the BLOSUM62matrix, a gap existence penalty of 11, and a gap extension penalty of 1.In some examples isolated polynucleotide encodes a polypeptide selectedfrom the group consisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or1240-1243. In some examples the isolated polynucleotides comprise apolynucleotide sequence of SEQ ID NO: 434-832, 1214-1221, 1234-1239, or1244-1247, or the complementary polynucleotide thereof.

In some examples the isolated polynucleotides encode an SuR polypeptidecomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-29 (SEQ ID NO: 10) to generate a BLAST bitscore of at least 200, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, or 750, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the isolatedpolynucleotides encode an SuR polypeptide comprising an amino acidsequence that can be optimally aligned with a polypeptide sequence ofL1-29 (SEQ ID NO: 10) to generate a BLAST bit score of at least 393,wherein the BLAST alignment used the BLOSUM62 matrix, a gap existencepenalty of 11, and a gap extension penalty of 1. In some examples theisolated polynucleotides encode SuR polypeptides comprising an aminoacid sequence that can be optimally aligned with a polypeptide sequenceof L1-29 (SEQ ID NO: 10) to generate a percent sequence identity of atleast 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity,wherein the sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the percent sequence identity isdetermined using a global alignment method using the GAP algorithm withdefault parameters for an amino acid sequence % identity and %similarity using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix. In some examples the isolated polynucleotidesencode SuR polypeptides comprising an amino acid sequence that can beoptimally aligned with a polypeptide sequence of L1-29 (SEQ ID NO: 10)to generate a BLAST similarity score of at least 400, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850, 900, 910,920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040,1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160,1170, 1180, 1190, or 1200 wherein the BLAST alignment used the BLOSUM62matrix, a gap existence penalty of 11, and a gap extension penalty of 1.In some examples the isolated polynucleotides encode SuR polypeptidescomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-29 (SEQ ID NO: 10) to generate a BLASTsimilarity score of at least 1006, wherein the BLAST alignment used theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L1-29 (SEQ ID NO: 10) to generatea BLAST e-value score of at least e-60, e-70, e-75, e-80, e-85, e-90,e-95, e-100, e-105, e-106, e-107, e-108, e-109, e-110, e-111, e-112,e-113, e-114, e-115, e-116, e-117, e-118, e-119, e-120, or e-125,wherein the BLAST alignment used the BLOSUM62 matrix, a gap existencepenalty of 11, and a gap extension penalty of 1. In some examplesisolated polynucleotide encodes a polypeptide is selected from the groupconsisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or 1240-1243. Insome examples the isolated polynucleotides comprise a polynucleotidesequence of SEQ ID NO: 434-832, 1214-1221, 1234-1239, or 1244-1247, orthe complementary polynucleotide thereof.

In some examples the isolated polynucleotides encode an SuR polypeptidecomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-02 (SEQ ID NO: 3) to generate a BLAST bitscore of at least 200, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, or 750, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the isolatedpolynucleotides encode SuR polypeptides comprising an amino acidsequence that can be optimally aligned with a polypeptide sequence ofL1-02 (SEQ ID NO: 3) to generate a percent sequence identity of at least50% 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, whereinthe sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the percent sequence identity isdetermined using a global alignment method using the GAP algorithm withdefault parameters for an amino acid sequence % identity and %similarity using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix. In some examples the isolated polynucleotidesencode SuR polypeptides comprising an amino acid sequence that can beoptimally aligned with a polypeptide sequence of L1-02 (SEQ ID NO: 3) togenerate a BLAST similarity score of at least 400, 425, 450, 475, 500,525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850, 900, 910, 920,930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050,1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170,1180, 1190, or 1200 wherein the BLAST alignment used the BLOSUM62matrix, a gap existence penalty of 11, and a gap extension penalty of 1.In some examples the isolated polynucleotides encode SuR polypeptidescomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-02 (SEQ ID NO: 3) to generate a BLAST e-valuescore of at least e-60, e-70, e-75, e-80, e-85, e-90, e-95, e-100,e-105, e-106, e-107, e-108, e-109, e-110, e-111, e-112, e-113, e-114,e-115, e-116, e-117, e-118, e-119, e-120, or e-125, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the isolatedpolynucleotides encode SuR polypeptides comprising an amino acidsequence that can be optimally aligned with a polypeptide sequence ofL1-02 (SEQ ID NO: 3) to generate a BLAST e-value score of at leaste-112, wherein the BLAST alignment used the BLOSUM62 matrix, a gapexistence penalty of 11, and a gap extension penalty of 1. In someexamples isolated polynucleotide encodes a polypeptide is selected fromthe group consisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or1240-1243. In some examples the isolated polynucleotides comprise apolynucleotide sequence of SEQ ID NO: 434-832, 1214-1221, 1234-1239, or1244-1247, or the complementary polynucleotide thereof.

In some examples the isolated polynucleotides encode an SuR polypeptidecomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-07 (SEQ ID NO: 4) to generate a BLAST bitscore of at least 200, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, or 750, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the isolatedpolynucleotides encode an SuR polypeptide comprising an amino acidsequence that can be optimally aligned with a polypeptide sequence ofL1-07 (SEQ ID NO: 4) to generate a BLAST bit score of at least 388,wherein the BLAST alignment used the BLOSUM62 matrix, a gap existencepenalty of 11, and a gap extension penalty of 1. In some examples theisolated polynucleotides encode SuR polypeptides comprising an aminoacid sequence that can be optimally aligned with a polypeptide sequenceof L1-07 (SEQ ID NO: 4) to generate a percent sequence identity of atleast 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity,wherein the sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L1-07 (SEQ ID NO: 4) to generatea percent sequence identity of at least 93% sequence identity, whereinthe sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the percent sequence identity isdetermined using a global alignment method using the GAP algorithm withdefault parameters for an amino acid sequence % identity and %similarity using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix. In some examples the isolated polynucleotidesencode SuR polypeptides comprising an amino acid sequence that can beoptimally aligned with a polypeptide sequence of L1-07 (SEQ ID NO: 4) togenerate a BLAST similarity score of at least 400, 425, 450, 475, 500,525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850, 900, 910, 920,930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050,1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170,1180, 1190, or 1200, wherein the BLAST alignment used the BLOSUM62matrix, a gap existence penalty of 11, and a gap extension penalty of 1.In some examples the isolated polynucleotides encode SuR polypeptidescomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-07 (SEQ ID NO: 4) to generate a BLASTsimilarity score of at least 996 wherein the BLAST alignment used theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L1-07 (SEQ ID NO: 4) to generatea BLAST e-value score of at least e-60, e-70, e-75, e-80, e-85, e-90,e-95, e-100, e-105, e-106, e-107, e-108, e-109, e-110, e-111, e-112,e-113, e-114, e-115, e-116, e-117, e-118, e-119, e-120, or e-125,wherein the BLAST alignment used the BLOSUM62 matrix, a gap existencepenalty of 11, and a gap extension penalty of 1. In some examples theisolated polynucleotides encode SuR polypeptides comprising an aminoacid sequence that can be optimally aligned with a polypeptide sequenceof L1-07 (SEQ ID NO: 4) to generate a BLAST e-value score of at leaste-111, wherein the BLAST alignment used the BLOSUM62 matrix, a gapexistence penalty of 11, and a gap extension penalty of 1. In someexamples isolated polynucleotide encodes a polypeptide is selected fromthe group consisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or1240-1243. In some examples the isolated polynucleotides comprise apolynucleotide sequence of SEQ ID NO: 434-832, 1214-1221, 1234-1239, or1244-1247, or the complementary polynucleotide thereof.

In some examples the isolated polynucleotides encode an SuR polypeptidecomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-20 (SEQ ID NO: 6) to generate a BLAST bitscore of at least 200, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, or 750, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the isolatedpolynucleotides encode SuR polypeptides comprising an amino acidsequence that can be optimally aligned with a polypeptide sequence ofL1-20 (SEQ ID NO: 6) to generate a percent sequence identity of at least50% 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, whereinthe sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L1-20 (SEQ ID NO: 6) to generatea percent sequence identity of at least 93% sequence identity, whereinthe sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the percent sequence identity isdetermined using a global alignment method using the GAP algorithm withdefault parameters for an amino acid sequence % identity and %similarity using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix. In some examples the isolated polynucleotidesencode SuR polypeptides comprising an amino acid sequence that can beoptimally aligned with a polypeptide sequence of L1-20 (SEQ ID NO: 6) togenerate a BLAST similarity score of at least 400, 425, 450, 475, 500,525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850, 900, 910, 920,930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050,1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170,1180, 1190, or 1200, wherein the BLAST alignment used the BLOSUM62matrix, a gap existence penalty of 11, and a gap extension penalty of 1.In some examples the isolated polynucleotides encode SuR polypeptidescomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-20 (SEQ ID NO: 6) to generate a BLAST e-valuescore of at least e-60, e-70, e-75, e-80, e-85, e-90, e-95, e-100,e-105, e-106, e-107, e-108, e-109, e-110, e-111, e-112, e-113, e-114,e-115, e-116, e-117, e-118, e-119, e-120, or e-125, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the isolatedpolynucleotides encode SuR polypeptides comprising an amino acidsequence that can be optimally aligned with a polypeptide sequence ofL1-20 (SEQ ID NO: 6) to generate a BLAST e-value score of at leaste-111, wherein the BLAST alignment used the BLOSUM62 matrix, a gapexistence penalty of 11, and a gap extension penalty of 1. In someexamples isolated polynucleotide encodes a polypeptide is selected fromthe group consisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or1240-1243. In some examples the isolated polynucleotides comprise apolynucleotide sequence of SEQ ID NO: 434-832, 1214-1221, 1234-1239, or1244-1247, or the complementary polynucleotide thereof.

In some examples the isolated polynucleotides encode an SuR polypeptidecomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-44 (SEQ ID NO: 13) to generate a BLAST bitscore of at least 200, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, or 750, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the isolatedpolynucleotides encode SuR polypeptides comprising an amino acidsequence that can be optimally aligned with a polypeptide sequence ofL1-44 (SEQ ID NO: 13) to generate a percent sequence identity of atleast 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity,wherein the sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L1-44 (SEQ ID NO: 13) to generatea percent sequence identity of at least 93% sequence identity, whereinthe sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the percent sequence identity isdetermined using a global alignment method using the GAP algorithm withdefault parameters for an amino acid sequence % identity and %similarity using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix. In some examples the isolated polynucleotidesencode SuR polypeptides comprising an amino acid sequence that can beoptimally aligned with a polypeptide sequence of L1-44 (SEQ ID NO: 13)to generate a BLAST similarity score of at least 400, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850, 900, 910,920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040,1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160,1170, 1180, 1190, or 1200, wherein the BLAST alignment used the BLOSUM62matrix, a gap existence penalty of 11, and a gap extension penalty of 1.In some examples the isolated polynucleotides encode SuR polypeptidescomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L1-44 (SEQ ID NO: 13) to generate a BLASTe-value score of at least e-60, e-70, e-75, e-80, e-85, e-90, e-95,e-100, e-105, e-106, e-107, e-108, e-109, e-110, e-111, e-112, e-113,e-114, e-115, e-116, e-117, e-118, e-119, e-120, or e-125, wherein theBLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 11,and a gap extension penalty of 1. In some examples isolatedpolynucleotide encodes a polypeptide is selected from the groupconsisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or 1240-1243. Insome examples the isolated polynucleotides comprise a polynucleotidesequence of SEQ ID NO: 434-832, 1214-1221, 1234-1239, or 1244-1247, orthe complementary polynucleotide thereof.

In some examples the isolated polynucleotides encode an SuR polypeptidecomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L6-3A09 (SEQ ID NO: 1228) to generate a BLASTbit score of at least 200, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, 525, 550, 575, 600, 625, 650, 675, 700, or 750, wherein theBLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 11,and a gap extension penalty of 1. In some examples the isolatedpolynucleotides encode an SuR polypeptide comprising an amino acidsequence that can be optimally aligned with a polypeptide sequence ofL6-3A09 (SEQ ID NO: 1228) to generate a BLAST bit score of at least 381,wherein the BLAST alignment used the BLOSUM62 matrix, a gap existencepenalty of 11, and a gap extension penalty of 1. In some examples theisolated polynucleotides encode SuR polypeptides comprising an aminoacid sequence that can be optimally aligned with a polypeptide sequenceof L6-3A09 (SEQ ID NO: 1228) to generate a percent sequence identity ofat least 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity,wherein the sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the percent sequence identity isdetermined using a global alignment method using the GAP algorithm withdefault parameters for an amino acid sequence % identity and %similarity using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix. In some examples the isolated polynucleotidesencode SuR polypeptides comprising an amino acid sequence that can beoptimally aligned with a polypeptide sequence of L6-3A09 (SEQ ID NO:1228) to generate a BLAST similarity score of at least 400, 425, 450,475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850, 900,910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030,1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150,1160, 1170, 1180, 1190, or 1200, wherein the BLAST alignment used theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L6-3A09 (SEQ ID NO: 1228) togenerate a BLAST similarity score of at least 978, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the isolatedpolynucleotides encode SuR polypeptides comprising an amino acidsequence that can be optimally aligned with a polypeptide sequence ofL6-3A09 (SEQ ID NO: 1228) to generate a BLAST e-value score of at leaste-60, e-70, e-75, e-80, e-85, e-90, e-95, e-100, e-105, e-106, e-107,e-108, e-109, e-110, e-111, e-112, e-113, e-114, e-115, e-116, e-117,e-118, e-119, e-120, or e-125, wherein the BLAST alignment used theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L6-3A09 (SEQ ID NO: 1228) togenerate a BLAST e-value score of at least e-108, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples isolated polynucleotideencodes a polypeptide is selected from the group consisting of SEQ IDNO: 3-401, 1206-1213, 1228-1233, or 1240-1243. In some examples theisolated polynucleotides comprise a polynucleotide sequence of SEQ IDNO: 434-832, 1214-1221, 1234-1239, or 1244-1247, or the complementarypolynucleotide thereof.

In some examples the isolated polynucleotides encode an SuR polypeptidecomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L6-3H02 (SEQ ID NO: 94) to generate a BLAST bitscore of at least 200, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, or 750, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the isolatedpolynucleotides encode SuR polypeptides comprising an amino acidsequence that can be optimally aligned with a polypeptide sequence ofL6-3H02 (SEQ ID NO: 94) to generate a percent sequence identity of atleast 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity,wherein the sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L6-3H02 (SEQ ID NO: 94) togenerate a percent sequence identity of at least 90% sequence identity,wherein the sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the percent sequence identity isdetermined using a global alignment method using the GAP algorithm withdefault parameters for an amino acid sequence % identity and %similarity using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix. In some examples the isolated polynucleotidesencode SuR polypeptides comprising an amino acid sequence that can beoptimally aligned with a polypeptide sequence of L6-3H02 (SEQ ID NO: 94)to generate a BLAST similarity score of at least 400, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850, 900, 910,920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040,1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160,1170, 1180, 1190, or 1200, wherein the BLAST alignment used the BLOSUM62matrix, a gap existence penalty of 11, and a gap extension penalty of 1.In some examples the isolated polynucleotides encode SuR polypeptidescomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L6-3H02 (SEQ ID NO: 94) to generate a BLASTe-value score of at least e-60, e-70, e-75, e-80, e-85, e-90, e-95,e-100, e-105, e-106, e-107, e-108, e-109, e-110, e-111, e-112, e-113,e-114, e-115, e-116, e-117, e-118, e-119, e-120, or e-125, wherein theBLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 11,and a gap extension penalty of 1. In some examples isolatedpolynucleotide encodes a polypeptide is selected from the groupconsisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or 1240-1243. Insome examples the isolated polynucleotides comprise a polynucleotidesequence of SEQ ID NO: 434-832, 1214-1221, 1234-1239, or 1244-1247, orthe complementary polynucleotide thereof.

In some examples the isolated polynucleotides encode an SuR polypeptidecomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L7-4E03 (SEQ ID NO: 1229) to generate a BLASTbit score of at least 200, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, 525, 550, 575, 600, 625, 650, 675, 700, or 750, wherein theBLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 11,and a gap extension penalty of 1. In some examples the isolatedpolynucleotides encode an SuR polypeptide comprising an amino acidsequence that can be optimally aligned with a polypeptide sequence ofL7-4E03 (SEQ ID NO: 1229) to generate a BLAST bit score of at least 368,wherein the BLAST alignment used the BLOSUM62 matrix, a gap existencepenalty of 11, and a gap extension penalty of 1. In some examples theisolated polynucleotides encode SuR polypeptides comprising an aminoacid sequence that can be optimally aligned with a polypeptide sequenceof L7-4E03 (SEQ ID NO: 1229) to generate a percent sequence identity ofat least 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity,wherein the sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the percent sequence identity isdetermined using a global alignment method using the GAP algorithm withdefault parameters for an amino acid sequence % identity and %similarity using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix. In some examples the isolated polynucleotidesencode SuR polypeptides comprising an amino acid sequence that can beoptimally aligned with a polypeptide sequence of L7-4E03 (SEQ ID NO:1229) to generate a BLAST similarity score of at least 400, 425, 450,475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850, 900,910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030,1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150,1160, 1170, 1180, 1190, or 1200, wherein the BLAST alignment used theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L7-4E03 (SEQ ID NO: 1229) togenerate a BLAST similarity score of at least 945, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the isolatedpolynucleotides encode SuR polypeptides comprising an amino acidsequence that can be optimally aligned with a polypeptide sequence ofL7-4E03 (SEQ ID NO: 1229) to generate a BLAST e-value score of at leaste-60, e-70, e-75, e-80, e-85, e-90, e-95, e-100, e-105, e-106, e-107,e-108, e-109, e-110, e-111, e-112, e-113, e-114, e-115, e-116, e-117,e-118, e-119, e-120, or e-125, wherein the BLAST alignment used theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L7-4E03 (SEQ ID NO: 1229) togenerate a BLAST e-value score of at least e-105, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples isolated polynucleotideencodes a polypeptide is selected from the group consisting of SEQ IDNO: 3-401, 1206-1213, 1228-1233, or 1240-1243. In some examples theisolated polynucleotides comprise a polynucleotide sequence of SEQ IDNO: 434-832, 1214-1221, 1234-1239, or 1244-1247, or the complementarypolynucleotide thereof.

In some examples the isolated polynucleotides encode an SuR polypeptidecomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L10-84(B12) (SEQ ID NO: 1230) to generate aBLAST bit score of at least 200, 250, 275, 300, 325, 350, 375, 400, 425,450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, or 750, whereinthe BLAST alignment used the BLOSUM62 matrix, a gap existence penalty of11, and a gap extension penalty of 1. In some examples the isolatedpolynucleotides encode SuR polypeptides comprising an amino acidsequence that can be optimally aligned with a polypeptide sequence ofL10-84(B12) (SEQ ID NO: 1230) to generate a percent sequence identity ofat least 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity,wherein the sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L10-84(B12) (SEQ ID NO: 1230) togenerate a percent sequence identity of at least 86% sequence identity,wherein the sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the percent sequence identity isdetermined using a global alignment method using the GAP algorithm withdefault parameters for an amino acid sequence % identity and %similarity using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix. In some examples the isolated polynucleotidesencode SuR polypeptides comprising an amino acid sequence that can beoptimally aligned with a polypeptide sequence of L10-84(B12) (SEQ ID NO:1230) to generate a BLAST similarity score of at least 400, 425, 450,475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850, 900,910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030,1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150,1160, 1170, 1180, 1190, or 1200, wherein the BLAST alignment used theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L10-84(B12) (SEQ ID NO: 1230) togenerate a BLAST e-value score of at least e-60, e-70, e-75, e-80, e-85,e-90, e-95, e-100, e-105, e-106, e-107, e-108, e-109, e-110, e-111,e-112, e-113, e-114, e-115, e-116, e-117, e-118, e-119, e-120, or e-125,wherein the BLAST alignment used the BLOSUM62 matrix, a gap existencepenalty of 11, and a gap extension penalty of 1. In some examplesisolated polynucleotide encodes a polypeptide is selected from the groupconsisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or 1240-1243. Insome examples the isolated polynucleotides comprise a polynucleotidesequence of SEQ ID NO: 434-832, 1214-1221, 1234-1239, or 1244-1247, orthe complementary polynucleotide thereof.

In some examples the isolated polynucleotides encode an SuR polypeptidecomprising an amino acid sequence that can be optimally aligned with apolypeptide sequence of L13-46 (SEQ ID NO: 1231) to generate a BLAST bitscore of at least 200, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, or 750, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the isolatedpolynucleotides encode an SuR polypeptide comprising an amino acidsequence that can be optimally aligned with a polypeptide sequence ofL13-46 (SEQ ID NO: 1231) to generate a BLAST bit score of at least 320,wherein the BLAST alignment used the BLOSUM62 matrix, a gap existencepenalty of 11, and a gap extension penalty of 1. In some examples theisolated polynucleotides encode SuR polypeptides comprising an aminoacid sequence that can be optimally aligned with a polypeptide sequenceof L13-46 (SEQ ID NO: 1231) to generate a percent sequence identity ofat least 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity,wherein the sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L13-46 (SEQ ID NO: 1231) togenerate a percent sequence identity of at least 86% sequence identity,wherein the sequence identity is determined by BLAST alignment using theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the percent sequence identity isdetermined using a global alignment method using the GAP algorithm withdefault parameters for an amino acid sequence % identity and %similarity using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix. In some examples the isolated polynucleotidesencode SuR polypeptides comprising an amino acid sequence that can beoptimally aligned with a polypeptide sequence of L13-46 (SEQ ID NO:1231) to generate a BLAST similarity score of at least 400, 425, 450,475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850, 900,910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030,1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150,1160, 1170, 1180, 1190, or 1200, wherein the BLAST alignment used theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L13-46 (SEQ ID NO: 1231) togenerate a BLAST similarity score of at least 819, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples the isolatedpolynucleotides encode SuR polypeptides comprising an amino acidsequence that can be optimally aligned with a polypeptide sequence ofL13-46 (SEQ ID NO: 1231) to generate a BLAST e-value score of at leaste-60, e-70, e-75, e-80, e-85, e-90, e-95, e-100, e-105, e-106, e-107,e-108, e-109, e-110, e-111, e-112, e-113, e-114, e-115, e-116, e-117,e-118, e-119, e-120, or e-125, wherein the BLAST alignment used theBLOSUM62 matrix, a gap existence penalty of 11, and a gap extensionpenalty of 1. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence that can be optimallyaligned with a polypeptide sequence of L13-46 (SEQ ID NO: 1231) togenerate a BLAST e-value score of at least e-90, wherein the BLASTalignment used the BLOSUM62 matrix, a gap existence penalty of 11, and agap extension penalty of 1. In some examples isolated polynucleotideencodes a polypeptide is selected from the group consisting of SEQ IDNO: 3-401, 1206-1213, 1228-1233, or 1240-1243. In some examples theisolated polynucleotides comprise a polynucleotide sequence of SEQ IDNO: 434-832, 1214-1221, 1234-1239, or 1244-1247, or the complementarypolynucleotide thereof.

In some examples the isolated polynucleotides encode an SuR polypeptidecomprising a ligand binding domain from a polypeptide selected from thegroup consisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or1240-1243. In some examples the isolated polynucleotides encode SuRpolypeptides comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or 1240-1243. Insome examples the encoded SuR polypeptide is selected from the groupconsisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, or 1240-1243, andthe sulfonylurea compound is selected from the group consisting ofchlorsulfuron, ethametsulfuron-methyl, metsulfuron-methyl,sulfometuron-methyl, and thifensulfuron-methyl. In some examples theisolated polynucleotides comprise a polynucleotide sequence of SEQ IDNO: 434-832, 1214-1221, 1234-1239, or 1244-1247, or the complementarypolynucleotide thereof.

In some examples the isolated SuR polynucleotide encodes an SuRpolypeptide having an equilibrium binding constant for a sulfonylureacompound greater than 0.1 nM and less than 10 μM. In some examples theencoded SuR polypeptide has an equilibrium binding constant for asulfonylurea compound of at least 0.1 nM, 0.5 nM, 1 nM, 10 nM, 50 nM,100 nM, 250 nM, 500 nM, 750 nM, 1 μM, 5 μM, 7 μM but less than 10 μM. Insome examples the encoded SuR polypeptide has an equilibrium bindingconstant for a sulfonylurea compound of at least 0.1 nM, 0.5 nM, 1 nM,10 nM, 50 nM, 100 nM, 250 nM, 500 nM, 750 nM but less than 1 μM. In someexamples the encoded SuR polypeptide has an equilibrium binding constantfor a sulfonylurea compound greater than 0 nM, but less than 0.1 nM, 0.5nM, 1 nM, 10 nM, 50 nM, 100 nM, 250 nM, 500 nM, 750 nM, 1 μM, 5 μM, 7μM, or 10 μM. In some examples the sulfonylurea compound is achlorsulfuron, an ethametsulfuron, a metsulfuron, a sulfometuron, and/ora thifensulfuron compound.

In some examples the isolated SuR polynucleotide encodes an SuRpolypeptide having an equilibrium binding constant for an operatorsequence greater than 0.1 nM and less than 10 μM. In some examples theencoded SuR polypeptide has an equilibrium binding constant for anoperator sequence of at least 0.1 nM, 0.5 nM, 1 nM, 10 nM, 50 nM, 100nM, 250 nM, 500 nM, 750 nM, 1 μM, 5 μM, 7 μM but less than 10 μM. Insome examples the encoded SuR polypeptide has an equilibrium bindingconstant for an operator sequence of at least 0.1 nM, 0.5 nM, 1 nM, 10nM, 50 nM, 100 nM, 250 nM, 500 nM, 750 nM but less than 1 μM. In someexamples the encoded SuR polypeptide has an equilibrium binding constantfor an operator sequence greater than 0 nM, but less than 0.1 nM, 0.5nM, 1 nM, 10 nM, 50 nM, 100 nM, 250 nM, 500 nM, 750 nM, 1 μM, 5 μM, 7 μMor 10 μM. In some examples the operator sequence is a Tet operatorsequence. In some examples the Tet operator sequence is a TetR(A)operator sequence, a TetR(B) operator sequence, a TetR(D) operatorsequence, TetR(E) operator sequence, a TetR(H) operator sequence or afunctional derivative thereof.

In some examples the isolated polynucleotides encoding SuR polypeptidescomprise codon composition profiles representative of codon preferencesfor particular host cells, or host cell organelles. In some examples theisolated polynucleotides comprise prokaryote preferred codons. In someexamples the isolated polynucleotides comprise bacteria preferredcodons. In some examples the bacteria is E. coli or Agrobacterium. Insome examples the isolated polynucleotides comprise plastid preferredcodons. In some examples the isolated polynucleotides comprise eukaryotepreferred codons. In some examples the isolated polynucleotides comprisenuclear preferred codons. In some examples the isolated polynucleotidescomprise plant preferred codons. In some examples the isolatedpolynucleotides comprise monocotyledonous plant preferred codons. Insome examples the isolated polynucleotides comprise corn, rice, sorghum,barley, wheat, rye, switch grass, turf grass and/or oat preferredcodons. In some examples the isolated polynucleotides comprisedicotyledonous plant preferred codons. In some examples the isolatedpolynucleotides comprise soybean, sunflower, safflower, Brassica,alfalfa, Arabidopsis, tobacco and/or cotton preferred codons. In someexamples the isolated polynucleotides comprise yeast preferred codons.In some examples the isolated polynucleotides comprise mammalianpreferred codons. In some examples the isolated polynucleotides compriseinsect preferred codons.

Compositions also include isolated polynucleotides fully complementaryto a polynucleotide encoding an SuR polypeptide, expression cassettes,replicons, vectors, T-DNAs, DNA libraries, host cells, tissues and/ororganisms comprising the polynucleotides encoding the SuR polypeptidesand/or complements or derivatives thereof. In some examples a DNAlibrary comprising a population of polynucleotides which encode apopulation of SuR polypeptide variants is provided. In some examples thepolynucleotide is stably incorporated into a genome of the host cell,tissue and/or organism. In some examples the host cell is a prokaryote,including E. coli and Agrobacterium strains. In some examples the hostis a eukaryote, including for example yeast, insects, plants andmammals.

Methods using the compositions are further provided. In one examplemethods of regulating transcription of a polynucleotide of interest in ahost cell are provided, the methods comprising: providing a cellcomprising the polynucleotide of interest operably linked to a promotercomprising at least one tetracycline operator sequence; providing an SuRpolypeptide and, providing a sulfonylurea compound, thereby regulatingtranscription of the polynucleotide of interest. Any host cell can beused, including for example prokaryotic cells such as bacteria, andeukaryotic cells, including yeast, plant, insect, and mammalian cells.In some examples providing the SuR polypeptide comprises contacting thecell with an expression cassette comprising a promoter functional in thecell operably linked to a polynucleotide that encodes the SuRpolypeptide.

Methods for generating and selecting diversified libraries to produceadditional SuR polynucleotides, including polynucleotides encoding SuRpolypeptides with improved and/or enhanced characteristics, e.g.,altered binding constants for sulfonylurea compounds and/or the targetDNA operator sequence and/or increased stability, all based uponselection of a polynucleotide constituent of the library for the new orimproved activities are also provided. In some examples at least onelibrary or population of oligonucleotides designed to introduce sequencemodifications and/or diversity to a wild type or modified TetR ligandbinding domain polypeptide is provided. In some examples the library orpopulation is designed to introduce modifications and/or diversity to awild type or modified TetR polypeptide. In some examples, the library orpopulation introduce at least one modification as exemplified in FIG. 9.In some examples the library or population comprises at least 10, 20,30, 40, 50, 60, 70, 80, 90, 100, or greater distinct oligonucleotides.In some examples the library or population comprises oligonucleotidesselected from the oligonucleotides shown in at least one of Table 2, 9,12, 13, 15, 17, 19 or a combination thereof. In some examples thelibrary or population comprises one or more oligonucleotides selectedfrom the group consisting of SEQ ID NO: 833-882, 885-986, 987-059,1060-1083, 1084-1124, 1125-1154, 1159-1205.

In some examples the sulfonylurea compound is an ethametsulfuron. Insome examples the ethametsulfuron is provided at a concentration ofabout 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009,0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.15, 0.2,0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85,0.9, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 200 or500 μg/ml. In some examples the SuR polypeptide has a ligand bindingdomain having at least 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to an SuR polypeptide of SEQ ID NO: 205-401,1206-1213, or 1228-1233, wherein the sequence identity is determinedover the full length of the polypeptide using a global alignment method.In some examples the global alignment method is GAP, wherein the defaultparameters are for an amino acid sequence % identity and % similarityusing GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoringmatrix. In some examples the polypeptide has a ligand binding domainfrom a SuR polypeptide selected from the group consisting of SEQ ID NO:205-401, 1206-1213, 1228-1233, or 1240-1243. In some examples thepolypeptide is selected from the group consisting of SEQ ID NO: 205-401,1206-1213, 1228-1233, or 1240-1243. In some examples the polypeptide isencoded by a polynucleotide of SEQ ID NO: 636-832, 1214-1221, 1234-1239,or 1244-1247.

In some examples the sulfonylurea compound is chlorsulfuron. In someexamples the chlorsulfuron is provided at a concentration of about 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.15, 0.2, 0.25,0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9,0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0,7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 200 or 500μg/ml. In some examples the SuR polypeptide has a ligand binding domainhaving at least 50% 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to an SuR polypeptide of SEQ ID NO: 14-204, wherein thesequence identity is determined over the full length of the polypeptideusing a global alignment method. In some examples the global alignmentmethod is GAP, wherein the default parameters are for an amino acidsequence % identity and % similarity using GAP Weight of 8 and LengthWeight of 2 and the BLOSUM62 scoring matrix. In some examples thepolypeptide has a ligand binding domain from a SuR polypeptide selectedfrom the group consisting of SEQ ID NO: 14-204. In some examples thepolypeptide is selected from the group consisting of SEQ ID NO: 14-204.In some examples the polypeptide is encoded by a polynucleotide of SEQID NO: 445-635.

The ability to capture value in various seed markets will requiredevelopment of technology for controlling engineered trait distribution.One option is a trait deactivation/activation system using achemically-regulated gene switch. To date no such system exists, inlarge part because of the lack of relevant chemistry, for exampleagricultural-compatible and/or pharmaceutical-based chemistry, that canbe used as a ligand for a sensitive gene switch technology.

To develop an agricultural chemical-based ligand gene switch, TetR wasmodified using protein modeling, DNA shuffling, and a highly sensitivescreening mechanism to produce a repressor that specifically recognizessulfonylurea compounds. For agricultural applications, sulfonylureacompounds are phloem mobile and commercially available, therebyproviding a good basis for use as switch ligand chemistry. Followingthree rounds of modeling and DNA shuffling, repressors that recognize SUchemistry nearly as well as wild type TetR recognizes cognate inducersand yet are totally specific to sulfonylurea chemistry have beengenerated. These polypeptides comprise true sulfonylurea repressors(SuRs), which have been validated in planta using a newly developedtransient assay system to demonstrate functionality of the SuR switchsystem. While exemplified in an agricultural context, these methods andcompositions can be used in a wide variety of other settings andorganisms.

In general, a chemical switch system wherein the chemical usedpenetrates rapidly and is perceived by all cell types in the organism,but does not perturb any endogenous regulatory networks will be mostuseful. Other important aspects have to do with the behavior of thesensor component, for example the stringency of regulation and responsein the absence or presence of inducer. In general a switch system havingtight regulation of the “off” state in the absence of inducer and rapidand intense response in the presence of inducer is preferred.

The ability to reversibly turn genes on and off has great utility forthe analyses of gene expression and function, particularly for thosegenes whose products are toxic to the cell. A well characterized controlmechanism in prokaryotes involves repressor proteins binding to operatorDNA to prevent transcription initiation (Wray and Reznikoff (1983) JBacteriol 156:1188-1191) and repressor-regulated systems have beendeveloped for controlling expression, both in animals (Wirtz and Clayton(1995) Science 268:1179-1183; Deuschle et al. (1995) Mol Cell Biol15:1097-1914; Furth et al. (1994) Proc Natl Acad Sci USA 91:9032-9306;Gossen and Bujard (1992) Proc Natl Acad Sci USA 89:5547-5551 and Gossenet al. (1995) Science 268:1766-1769) and plants (Wilde et al. (1992)EMBO J 11:1251-1259; Gatz et al. (1992) Plant J 2:397-404; Roder et al.(1994) Mol Gen Genet. 243-32-38 and Ulmasov et al. (1997) Plant Mol Biol35:417-424).

Two major repressor based systems have been successfully exploited forregulation of plant gene expression: the lac operator-repressor system(Ulmasov et al. (1997) Plant Mol Biol 35:417-424; Wilde et al. (1992)EMBO J 11:1251-1259) and the tet operator-repressor system (Wilde et al.(1992) EMBO J 11:1251-1259; Gatz et al. (1992) Plant J 2:397-404; Roderet al. (1994) Mol Gen Genet 243:32-38; Ulmasov et al. (1997) Plant MolBiol 35-417-424). Both are repressor/operator based-systems deriving keyelements from their corresponding prokaryotic operon, namely the E. colilactose operon for lac and the transposon Tn10 tetracycline operon fortet. Generally, these systems control the activity of a promoter byplacing operator sequences near the transcriptional start site of a genesuch that gene expression from the operon is inhibited upon the bindingof the repressor protein to its cognate operator sequence. However, inthe presence of an inducing agent, the binding of the repressor to itsoperator is inhibited, thus activating the promoter and enabling geneexpression. In the lac system, isopropyl-B-D-thiogalactopyranoside(IPTG) is the commonly used inducing agent, while tetracycline and/ordoxycycline are commonly used inducing agents for the tet system.

Expression of the Tn10-operon is regulated by binding of the tetrepressor to its operator sequences (Beck et al. (1982) J Bacteriol150:633-642; Wray and Reznikoff (1983) J Bacteriol 156:1188-1191). Thehigh specificity of tetracycline repressor for the tet operator, thehigh efficiency of induction by tetracycline and its derivatives, thelow toxicity of the inducer, as well as the ability of tetracycline toeasily permeate most cells, are the basis for the application of the tetsystem in somatic gene regulation in eukaryotic cells from animals(Wirtz and Clayton (1995) Science 268:1179-1183; Gossen et al. (1995)Science 268:1766-1769), humans (Deuschle et al. (1995) Mol Cell Biol15:1907-1914; Furth et al. (1994) Proc Natl Acad Sci USA 91:9302-9306;Gossen and Bujard (1992) Proc Natl Acad Sci USA 89:5547-5551; Gossen etal. (1995) Science 268:1766-1769) and plant cell cultures (Wilde et al.(1992) EMBO J 11:1251-1259; Gatz et al. (1992) Plant J 2:397-404; Roderet al. (1994) Mol Gen Genet 243:32-28; Ulmasov et al. (1997) Plant MolBiol 35:417-424).

A number of variations of tetracycline operator/repressor systems havebeen devised. For example, one system based on conversion of the tetrepressor to an activator was developed via fusion of the repressor to atranscriptional transactivation domain such as herpes simplex virus VP16and the tet repressor (tTA, Gossen and Bujard (1992) Proc Natl Acad SciUSA 89:5547-5551). In this system, a minimal promoter is activated inthe absence of tetracycline by binding of tTA to tet operator sequences,and tetracycline inactivates the transactivator and inhibitstranscription. This system has been used in plants (Weinmann et al.(1994) Plant J 5:559-569), rat hearts (Fishman et al. (1994) J ClinInvest 93:1864-1868) and mice (Furth et al. (1994) Proc Natl Acad SciUSA 91:9302-9306). However, there were indications that the chimeric tTAfusion protein was toxic to cells at levels required for efficient generegulation (Bohl et al. (1996) Nat Med 3:299-305).

Promoters modified to be regulated by tetracycline and analogs thereofare known (Matzke et al. (2003) Plant Mol Biol Rep 21:9-19; Padidam(2003) Curr Op Plant Biol 6:169-177; Gatz and Quail (1988) Proc NatlAcad Sci USA 85:1394-1397; Ulmasov et al. (1997) Plant Mol Biol35:417-424; Weinmann, et al. (1994) Plant J 5:559-569). One or more tetoperator sequences can be added to a promoter in order to produce atetracycline inducible promoter. In some examples up to 7 tet operatorshave been introduced upstream of a minimal promoter sequence and aTetR::VP16 activation domain fusion applied in trans activatesexpression only in the absence of inducer (Weinmann et al. (1994) PlantJ 5:559-569; Love et al. (2000) Plant J 21:579-588). A widely testedtetracycline regulated expression system for plants using the CaMV 35Spromoter was developed (Gatz et al. (1992) Plant J 2:397-404) havingthree tet operators introduced near the TATA box (3XOpT 35S). The 3XOpT35S promoter generally functioned in tobacco and potato, howevertoxicity and poor plant phenotype in tomato and Arabidopsis (Gatz (1997)Ann Rev Plant Physiol Plant Mol Biol 48:89-108; Corlett et al. (1996)Plant Cell Environ 19:447-454) were also reported. Another factor isthat the tetracycline-related chemistry is rapidly degraded in thelight, which tends to confine its use to testing in laboratoryconditions.

TetR has been subjected to DNA shuffling to modify its inducerspecificity from tetracycline to4-de(dimethylamino)-6-deoxy-6-demethyl-tetracycline (cmt3) a related butnon-inducing compound (Scholz et al. (2003) J Mol Biol 329:217-227)which lacks chemical side groups at positions 4 and 6 and is thereforesmaller than tetracycline. The specificity of TetR was altered bynarrowing the ligand binding pocket, thereby sterically blocking thenatural ligand tetracycline. The starting polypeptide was a TetR(BD)chimera consisting of amino acids 1-50 from TetR(B) and residues 51-208from TetR(D). Several rounds of evolution and selection were used toshift TetR specificity from tetracycline to cmt3. Non-inducer cmt3 hadlittle starting activity and was brought to the level of tetracycline,yielding an improvement in activity of several thousand-fold, andtetracycline has almost no inducing activity with the mutant repressors.While the ability to shift the specificity of TetR to the cmt3 ligand isexciting, it must be kept in mind that cmt3 is highly related to thenatural tetracycline ligand. Based on these experiments, it is notobvious that TetR could be used as the basis for developing specificityto a completely different class of chemical ligands.

To produce a new chemical switch system, we re-designed the TetR systemto recognize chemistry viable for use in agriculture. The re-designprocess was initiated by choosing a registered agrichemical compoundhaving excellent plant uptake and distribution properties, as well ashaving a size and a shape reasonable for modeling into the wild typeTetR ligand binding pocket. The compound chosen, thifensulfuron-methyl(Harmony®) is one of a family of commercially used sulfonylurea typeherbicides inhibiting the key plant enzyme in branched chain amino acidbiosynthesis, acetolactate synthase (ALS). Thifensulfuron (Ts) andrelated herbicides are structurally disparate to tetracycline, thereforeit was unlikely they would have any starting activity with TetR. DNAshuffling is a powerful technology and can improve affinities forsubstrates or rate of substrate turnover by several thousand-fold,however has not yet been able to create de novo starting activity. Tomeet this gap in the evolution pathway a computer modeling strategy wassought that would narrow the search for meaningful amino acid diversityfor shuffling. Recently developed modeling technology was used tore-train E. coli periplasmic binding proteins that normally bind tosugars to react to and initiate signaling with completely diverse setsof compounds such as serotonin, L-lactate and trinitrotoluene (Looger etal. (2003) Nature 423:185-190). Using protein design coupled with DNAshuffling and a very sensitive screening system, TetR protein variantsthat respond to thifensulfuron (Ts) and other related SU compounds havebeen identified. Following several rounds of DNA shuffling, TetRvariants were developed having genetic switch capability with SU ligands(SuRs) similar to that of TetR with tetracycline inducers.

Any method of rational protein design can be used alone or incombination. For example, phylogenetic diversity within a family ofprotein sequences can be used to identify positions in the primarystructure having amino acid substitutions, and the types ofsubstitutions that have occurred and their impact on function. Conserveddomain families can also be aligned and similarly examined to identifypositions in the primary structure having amino acid substitutions andthe types of substitutions that have occurred and the impact onfunction. The secondary structure(s) and functional domains can beevaluated and various models used to predict tolerance or impact ofamino acid substitutions on structure and function. Modeling using thetertiary and/or quaternary structure and ligand, substrate and/orcofactor binding provide further insights into the effects of amino acidsubstitutions and/or alternate ligands, substrates and/or cofactorsinteractions with the polypeptide.

To examine the phylogenetic diversity of tetracycline repressors, both abroad family of tetracycline repressor proteins as well as closelyrelated tetracycline repressors were used. Thirty-four proteins wereidentified and aligned to examine the amino acid diversity at variouspositions in the repressor family (SEQ ID NO: 1 and 402-433). The broadfamily of tetracycline repressors comprised a TetR(D) mutant whosestructure was determined by crystallization PDB_(—)1A6I (Orth et al.(1998) J Mol Biol 279:439-447) and public sequence deposit accessionsA26948, AAA98409, AAD12754, AAD25094, AAD25537, AAP93923, AAR96033,AAW66496, AAW83818, ABO14708, ABS19067, CAA24908, CAC80726, CAC81917,EAY62734, NP_(—)387455, NP_(—)387462, NP_(—)511232, NP_(—)824556,P51560, YP_(—)001220607, YP_(—)001370475, YP_(—)368094, YP_(—)620166,YP_(—)772551, ZP_(—)00132379, ZP_(—)01558383, and ZP_(—)01567051.Closely related tetracycline repressors included TetR(A) P03038, TetR(B)P04483, TetR(D) P0ACT4, TetR(E) P21337 and TetR(H) P51561. Thealignments of these sequences were used to look at overall sequencediversity as well as diversity in the DNA and the ligand binding domains(see, Example 1H, SEQ ID NO: 1 and 402-433).

The modular architecture of repressor proteins and the commonality ofhelix-turn-helix DNA binding domains allows for the creation of SuRpolypeptides having altered DNA binding specificity. For example, theDNA binding specificity can be altered by fusing a SuR ligand bindingdomain to an alternate DNA binding domain. For example, the DNA bindingdomain from TetR class D can be fused to an SuR ligand binding domain tocreate SuR polypeptides that specifically bind to polynucleotidescomprising a class D tetracycline operator. In some examples a DNAbinding domain variant or derivative can be used. For example, a DNAbinding domain from a TetR variant that specifically recognizes atetO-4C operator or a tetO-6C operator could be used (Helbl and Hillen(1998) J Mol Biol 276:313-318; HelbI et al. (1998) J Mol Biol276:319-324. The four helix bundle formed by helices α8 and α10 in bothsubunits can be substituted to ensure dimerization specificity whentargeting two different operator specific repressor variants in the samecell to prevent heterodimerization (e.g., Rossi et al. (1998) Nat Genet20:389-393; Berens and Hillen (2003) Eur J Biochem 270:3109-3121). Inanother example, the DNA binding domain from LexA repressor was fused toGAL4 wherein this hybrid protein recognized LexA operators in both E.coli and yeast (Brent and Ptashne (1985) Cell 43:729-736). In anotherexample, all of the presumptive DNA binding or DNA-recognition R-groupsof the 434 repressor were replaced by the corresponding positions of theP22 repressor. Operator binding specificity of the hybrid repressor434R[α3(P22R)] was tested both in vivo and in vitro and each test showedthat this targeted modification of 434 shifted the DNA bindingspecificity from 434 operator to P22 operator (Wharton and Ptashne(1985) Nature 316:601-605). This work was further extended by creating aheterodimer of wild type 434R and 434R[α3(P22R)] which then specificallyrecognized a chimeric P22/434 operator sequence (Hollis et al. (1988)Proc Natl Acad Sci USA 85:5834-5838). In another example, the N-terminalhalf of the AraC protein was fused to the LexA repressor DNA bindingdomain. The resulting AraC:LexA chimera dimerized, bound LexA operator,and repressed expression of a LexA operator:β-galactosidase fusion genein an arabinose-responsive manner (Bustos and Schleif (1993) Proc NatlAcad Sci USA 90:5638-5642).

The isolated polynucleotides encoding SuR polypeptides can also be usedas substrates for diversity-generating procedures, including mutation,recombination, and recursive recombination reactions, to produceadditional SuR polynucleotide and/or polypeptide variants with desiredproperties. Additionally, the SuR polynucleotides can be used fordiversity-generating procedures to produce polynucleotide and/orpolypeptide variants having an altered characteristic as compared to thestarting material, for example binding to a different ligand inducer.The diversity-generating process produces sequence alterations includingsingle nucleotide substitutions, multiple nucleotide substitutions andinsertion or deletion of regions of the nucleic acid sequence. Thediversity-generating procedures can be used separately and/or incombination to produce one or more SuR variants or set of variant aswell variants of encoded proteins. Individually and collectively, theseprocedures provide robust, widely applicable ways of generatingdiversified polynucleotides and polypeptides, as well as sets ofpolynucleotides and polypeptides, including, libraries. These variantsand sets of variants are useful for the engineering or rapid evolutionof polynucleotides, proteins, pathways, cells and/or organisms with newand/or improved characteristics. The resulting polynucleotide and/orpolypeptide variants can be selected or screened for alteredcharacteristics and/or properties, including altered ligand binding,retention of DNA binding, and/or quantification of binding properties.

Any method can be used to provide sequence diversity to a library. Manydiversity-generating procedures, including multigene shuffling andmethods for generating modified nucleic acid sequences are available,including for example, Soong et al. (2000) Nat Genet 25:436-39; Stemmeret al. (1999) Tumor Targeting 4:1-4; Ness et al. (1999) Nature Biotech17:893-896; Chang et al. (1999) Nature Biotech 17:793-797; Minshull andStemmer (1999) Curr Op Chem Biol 3:284-290; Christians et al. (1999)Nature Biotech 17:259-264; Crameri et al. (1998) Nature 391:288-291;Crameri et al. (1997) Nature Biotech 15:436-438; Zhang et al. (1997)Proc Natl Acad Sci USA 94:4504-4509; Patten et al. (1997) Curr OpBiotech 8:724-733; Crameri et al. (1996) Nature Med 2:100-103; Crameriet al. (1996) Nature Biotech 14:315-319; Gates et al. (1996) J Mol Biol255:373-386; Stemmer (1996) “Sexual PCR and Assembly PCR” in TheEncyclopedia of Molecular Biology (VCH Publishers, New York) pp.447-457; Crameri and Stemmer (1995) BioTechniques 18:194-195; Stemmer etal. (1995) Gene 164:49-53; Stemmer (1995) Science 270:1510; Stemmer(1995) Bio/Technology 13:549-553; Stemmer (1994) Nature 370:389-391 andStemmer (1994) Proc Natl Acad Sci USA 91:10747-10751. Mutational methodsto generate diversity include, for example, site-directed mutagenesis(Ling et al. (1997) Anal Biochem 254:157-178; Dale et al. (1996) MethodsMol Biol 57:369-374; Smith (1985) Ann Rev Genet 19:423-462; Botstein andShortle (1985) Science 229:1193-1201; Carter (1986) Biochem J 237:1-7and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids and Molecular Biology (Eckstein andLilley, eds., Springer Verlag, Berlin). Mutagenesis methods using uracilcontaining templates included Kunkel (1985) Proc Natl Acad Sci USA82:488-492; Kunkel et al. (1987) Methods Enzymol 154:367-382; and Basset al. (1988) Science 242:240-245. Oligonucleotide-directed mutagenesismethods include Zoller and Smith (1983) Methods Enzymol 100:468-500;Zoller and Smith (1982) Nucl Acids Res 10:6487-6500 and Zoller and Smith(1987) Methods Enzymol 154:329-350. Phosphorothioate-modified DNAmutagenesis methods include Taylor et al. (1985) Nucl Acids Res13:8749-8764; Taylor et al. (1985) Nucl Acids Res 13:8765-8787; Nakamayeand Eckstein (1986) Nucl Acids Res 14:9679-9698; Sayers et al. (1988)Nucl Acids Res 16:791-802 and Sayers et al. (1988) Nucl Acids Res16:803-814. Mutagenesis methods using gapped duplex DNA include (Krameret al. (1984) Nucl Acids Res 12:9441-9456; Kramer and Fritz (1987)Methods Enzymol 154:350-367; Kramer et al. (1988) Nucl Acids Res16:7207; and Fritz et al. (1988) Nucl Acids Res 16:6987-6999. Additionalsuitable diversity-generating methods include point mismatch repair(Kramer et al. (1984) Cell 38:879-887); mutagenesis usingrepair-deficient host strains (Carter et al. (1985) Nucl Acids Res13:4431-4443; and Carter (1987) Methods Enzymol 154:382-403); deletionmutagenesis (Eghtedarzadeh and Henikoff (1986) Nucl Acids Res 14: 5115);restriction-selection and restriction-purification (Wells et al. (1986)Phil Trans R Soc Lond A 317:415-423); mutagenesis by total genesynthesis (Nambiar et al. (1984) Science 223:1299-1301; Sakamar andKhorana (1988) Nucl Acids Res 14:6361-6372; Wells et al. (1985) Gene34:315-323 and Grundström et al. (1985) Nucl Acids Res. 13:3305-3316);double-strand break repair (Mandecki (1986) Proc Natl Acad Sci USA83:7177-7181; and Arnold (1993) Curr Op Biotech 4:450-455). Nucleicacids can be recombined in vitro by any technique or combination oftechniques including, e.g., DNAse digestion of nucleic acids to berecombined followed by ligation and/or PCR reassembly of the nucleicacids. For example, sexual PCR mutagenesis can be used in whichfragmentation of the DNA molecule is followed by recombination in vitro,based on sequence similarity, between DNA molecules with different butrelated DNA sequences, followed by fixation of the crossover byextension in a polymerase chain reaction. Similarly, nucleic acids canbe recursively recombined in vivo, e.g., by allowing recombination tooccur between nucleic acids in cells. Such formats optionally providedirect recombination between nucleic acids of interest, or providerecombination between constructs, vectors, viruses, and/or plasmidscomprising the nucleic acids of interest. Whole genome recombinationmethods can also be used wherein whole genomes of cells or otherorganisms are recombined, optionally including spiking of the genomicrecombination mixtures with desired library components. These methodshave many applications, including those in which the identity of atarget gene is not known. Any of these processes can be used alone or incombination to generate polynucleotides encoding SuR polypeptides. Anyof the diversity-generating methods can be used in a reiterativefashion, using one or more cycles of mutation/recombination or otherdiversity generation methods, optionally followed by one or moreselection methods to generate additional recombinant nucleic acids.

For convenience and high throughput it will often be desirable toscreen/select for desired modified nucleic acids in a microorganism,such as in a bacteria such as E. coli, or unicellular eukaryote such asyeast including S. cerevisiae, S. pombe, P. pastoris or protists such asChlamydomonas, or in model cell systems such as SF9, Hela, CHO, BMS,BY2, or other cell culture systems. In some instances, screening inplant cells or plants may be desirable, including plant cell or explantculture systems or model plant systems such as Arabidopsis, or tobacco.In some examples throughput is increased by screening pools of hostcells expressing different modified nucleic acids, either alone or aspart of a gene fusion construct. Any pools showing significant activitycan be deconvoluted to identify single clones expressing the desirableactivity.

Recombinant constructs comprising one or more of nucleic acid sequencesencoding a SuR polypeptide are provided. The constructs comprise avector, such as, a plasmid, a cosmid, a phage, a virus, a bacterialartificial chromosome (BAC), a yeast artificial chromosome (YAC), or thelike, into which a polynucleotide encoding a SuR polypeptide has beeninserted. In some examples, the construct further comprises regulatorysequences, including, for example, a promoter, operably linked to thesequence. Suitable vectors are well known and include chromosomal,non-chromosomal and synthetic DNA sequences, such as derivatives ofSV40; bacterial plasmids; replicons; phage DNA; baculovirus; yeastplasmids; vectors derived from combinations of plasmids and phage DNA,viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies,adenovirus, adeno-associated viruses, retroviruses, geminiviruses, TMV,PVX, other plant viruses, Ti plasmids, Ri plasmids and many others.

The vectors may optionally contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cells.Usually, the selectable marker gene will encode antibiotic or herbicideresistance. Suitable genes include those coding for resistance to theantibiotic spectinomycin or streptomycin (e.g., the aadA gene), thestreptomycin phosphotransferase (SPT) gene for streptomycin resistance,the neomycin phosphotransferase (NPTII or NPTIII) gene kanamycin orgeneticin resistance, the hygromycin phosphotransferase (HPT) gene forhygromycin resistance. Additional selectable marker genes includedihydrofolate reductase or neomycin resistance for eukaryotic cellculture and tetracycline or ampicillin resistance. Genes coding forresistance to herbicides include those which act to inhibit the actionof glutamine synthase, such as phosphinothricin or basta (e.g., the bargene), EPSPS, GOX, or GAT which provide resistance to glyphosate, mutantALS (acetolactate synthase) which provides resistance to sulfonylureatype herbicides or any other known genes.

In bacterial systems a number of expression vectors are available. Suchvectors include, but are not limited to, multifunctional E. coli cloningand expression vectors such as BLUESCRIPT (Stratagene); pIN vectors (VanHeeke and Schuster, (1989) J Biol Chem 264:5503-5509); pET vectors(Novagen, Madison Wis.) and the like. Similarly, in S. cerevisiae anumber of vectors containing constitutive or inducible promoters such asalpha factor, alcohol oxidase and PGH may be used for production ofpolypeptides. For reviews, see, Ausubel and Grant et al. (1987) MethEnzymol 153:516-544. A variety of expression systems can be used inmammalian host cells, including viral-based systems, such as adenovirusand rous sarcoma virus (RSV) systems. Any number of commercially orpublicly available expression systems or derivatives thereof can beused.

In plant cells expression can be driven from an expression cassetteintegrated into a plant chromosome, or an organelle, or cytoplasmicallyfrom an episomal or viral nucleic acid. Numerous plant derivedregulatory sequences have been described, including sequences whichdirect expression in a tissue specific manner, e.g., TobRB7, patatinB33, GRP gene promoters, the rbcS-3A promoter and the like.Alternatively, high level expression can be achieved by transientlyexpressing exogenous sequences of a plant viral vector, e.g., TMV, BMV,geminiviruses including WDV and the like.

Typical vectors useful for expression of nucleic acids in higher plantsare known including vectors derived from the tumor-inducing (Ti) plasmidof Agrobacterium tumefaciens described by Rogers et al. (1987) MethEnzymol 153:253-277. Exemplary A. tumefaciens vectors include plasmidspKYLX6 and pKYLX7 of Schardl et al. (1987) Gene 61:1-11 and Berger etal. (1989) Proc Natl Acad Sci USA 86:8402-8406 and plasmid pB101.2 thatis available from Clontech Laboratories, Inc. (Palo Alto, Calif.). Avariety of known plant viruses can be employed as vectors includingcauliflower mosaic virus (CaMV), geminiviruses, brome mosaic virus andtobacco mosaic virus.

The SuR may be used to control expression of a polynucleotide ofinterest. The polynucleotide of interest may be any sequence ofinterest, including but not limited to sequences encoding a polypeptide,encoding an mRNA, encoding an RNAi precursor, encoding an active RNAiagent, a miRNA, an antisense polynucleotide, a ribozyme, a fusionprotein, a replicating vector, a screenable marker, and the like.Expression of the polynucleotide of interest may be used to induceexpression of an encoding RNA and/or polypeptide, or conversely tosuppress expression of an encoded RNA, RNA target sequence, and/orpolypeptide. In specific examples, the polynucleotide sequence may apolynucleotide encoding a plant hormone, plant defense protein, anutrient transport protein, a biotic association protein, a desirableinput trait, a desirable output trait, a stress resistance gene, aherbicide resistance gene, a disease/pathogen resistance gene, a malesterility, a developmental gene, a regulatory gene, a DNA repair gene, atranscriptional regulatory gene or any other polynucleotide and/orpolypeptide of interest.

A number of promoters can be used in the compositions and methods. Forexample, a polynucleotide encoding a SuR polypeptide can be operablylinked to a constitutive, tissue-preferred, inducible, developmentally,temporally and/or spatially regulated or other promoters including thosefrom plant viruses or other pathogens which function in a plant cell. Avariety of promoters useful in plants is reviewed in Potenza et al.(2004) In Vitro Cell Dev Biol Plant 40:1-22.

Any polynucleotide, including polynucleotides of interest,polynucleotides encoding SuRs, regulatory regions, introns, promoters,and promoters comprising TetOp sequences may be obtained and theirnucleotide sequence determined, by any standard method. Thepolynucleotides may be chemically synthesized in their full-length orassembled from chemically synthesized oligonucleotides (Kutmeier et al.(1994) BioTechniques 17:242). Assembly from oligonucleotides typicallyinvolves synthesis of overlapping oligonucleotides, annealing andligating of those oligonucleotides and PCR amplification of the ligatedproduct. Alternatively, a polynucleotide may be isolated or generatedfrom a suitable source including suitable source a cDNA librarygenerated from tissue or cells, a genomic library, or directly isolatedfrom a host by PCR amplification using specific primers to the 3′ and 5′ends of the sequence or by cloning using an nucleotide probe specificfor the polynucleotide of interest. Amplified nucleic acid moleculesgenerated by PCR may then be cloned into replicable cloning vectorsusing standard methods. The polynucleotide may be further manipulatedusing any standard methods including recombinant DNA techniques, vectorconstruction, mutagenesis and PCR (see, e.g., Sambrook et al. (1990)Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.; Ausubel et al., Eds. (1998)Current Protocols in Molecular Biology, John Wiley and Sons, NY).

Any method for introducing a sequence into a cell or organism can beused, as long as the polynucleotide or polypeptide gains access to theinterior of at least one cell. Methods for introducing sequences intoplants are known and include, but are not limited to, stabletransformation, transient transformation, virus-mediated methods, andsexual breeding. Stably incorporated indicates that the introducedpolynucleotide is integrated into a genome and is capable of beinginherited by progeny. Transient transformation indicates that anintroduced sequence does not integrate into a genome such that it isheritable by progeny from the host. Any means can be used to bringtogether a SuR and polynucleotide of interest operably linked to apromoter comprising TetOp including, for example, stable transformation,transient delivery, cell fusion, sexual crossing or any combinationthereof.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell targeted for transformation. Suitablemethods of introducing polypeptides and polynucleotides into plant cellsinclude microinjection (Crossway et al. (1986) Biotechniques 4:320-334and U.S. Pat. No. 6,300,543), electroporation (Riggs et al. (1986) ProcNatl Acad Sci USA 83:5602-5606, Agrobacterium-mediated transformation(U.S. Pat. Nos. 5,563,055 and 5,981,840), direct gene transfer(Paszkowski et al. (1984) EMBO J 3:2717-2722), ballistic particleacceleration (U.S. Pat. Nos. 4,945,050, 5,879,918, 5,886,244 and5,932,782; Tomes et al. (1995) in Plant Cell, Tissue and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);McCabe et al. (1988) Biotechnology 6:923-926). Also see, Weissinger etal. (1988) Ann Rev Genet 22:421-477; Sanford et al. (1987) ParticulateScience and Technology 5:27-37; Christou et al. (1988) Plant Physiol87:671-674; Finer and McMullen (1991) In Vitro Cell Dev Biol 27P:175-182(soybean); Singh et al. (1998) Theor Appl Genet 96:319-324; Datta et al.(1990) Biotechnology 8:736-740; Klein et al. (1988) Proc Natl Acad SciUSA 85:4305-4309; Klein et al. (1988) Biotechnology 6:559-563; U.S. Pat.Nos. 5,240,855, 5,322,783 and 5,324,646; Klein et al. (1988) PlantPhysiol 91:440-444; Fromm et al. (1990) Biotechnology 8:833-839;Hooykaas-Van Slogteren et al. (1984) Nature 311:763-764; U.S. Pat. No.5,736,369; Bytebier et al. (1987) Proc Natl Acad Sci USA 84:5345-5349;De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues,ed. Chapman et al. (Longman, New York), pp. 197-209; Kaeppler et al.(1990) Plant Cell Rep 9:415-418; Kaeppler et al. (1992) Theor Appl Genet84:560-566; D'Halluin et al. (1992) Plant Cell 4:1495-1505; Li et al.(1993) Plant Cell Rep 12:250-255; Christou and Ford (1995) Ann Bot75:407-413 and Osjoda et al. (1996) Nat Biotechnol 14:745-750.Alternatively, polynucleotides may be introduced into plants bycontacting plants with a virus, or viral nucleic acids. Methods forintroducing polynucleotides into plants via viral DNA or RNA moleculesare known, see, e.g., U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785,5,589,367, 5,316,931 and Porta et al. (1996) Mol Biotech 5:209-221.

The term plant includes plant cells, plant protoplasts, plant celltissue cultures from which a plant can be regenerated, plant calli,plant clumps and plant cells that are intact in plants or parts ofplants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers and the like. Progeny, variants and mutants of the regeneratedplants are also included.

In some examples, a SuR may be introduced into a plastid, either bytransformation of the plastid or by directing a SuR transcript orpolypeptide into the plastid. Any method of transformation, nuclear orplastid, can be used, depending on the desired product and/or use.Plastid transformation provides advantages including high transgeneexpression, control of transgene expression, ability to expresspolycistronic messages, site-specific integration via homologousrecombination, absence of transgene silencing and position effects,control of transgene transmission via uniparental plastid geneinheritance and sequestration of expressed polypeptides in the organellewhich can obviate possible adverse impacts on cytoplasmic components(e.g., see, reviews including Heifetz (2000) Biochimie 82:655-666;Daniell et al. (2002) Trends Plant Sci 7:84-91; Maliga (2002) Curr OpPlant Biol 5:164-172; Maliga (2004) Ann Rev Plant Biol 55-289-313;Daniell et al. (2005) Trends Biotechnol 23:238-245 and Verma and Daniell(2007) Plant Physiol 145:1129-1143).

Methods and compositions of plastid transformation are well known, forexample, transformation methods include (Boynton et al. (1988) Science240:1534-1538; Svab et al. (1990) Proc Natl Acad Sci USA 87:8526-8530;Svab et al. (1990) Plant Mol Biol 14:197-205; Svab et al. (1993) ProcNatl Acad Sci USA 90:913-917; Golds et al. (1993) Bio/Technology11:95-97; O'Neill et al. (1993) Plant J 3:729-738; Koop et al. (1996)Planta 199:193-201; Kofer et al. (1998) In Vitro Plant 34:303-309;Knoblauch et al. (1999) Nat Biotechnol 17:906-909); as well as plastidtransformation vectors, elements, and selection (Newman et al. (1990)Genetics 126:875-888; Goldschmidt-Clermont, (1991) Nucl Acids Res19:4083-4089; Carrer et al. (1993) Mol Gen Genet 241:49-56; Svab et al.(1993) Proc Natl Acad Sci USA 90:913-917; Verma and Daniell (2007) PlantPhysiol 145:1129-1143).

Methods and compositions for controlling gene expression in plastids arewell known including (McBride et al. (1994) Proc Natl Acad Sci USA91:7301-7305; Lössl et al. (2005) Plant Cell Physiol 46:1462-1471;Heifetz (2000) Biochemie 82:655-666; Surzycki et al. (2007) Proc NatlAcad Sci USA 104:17548-17553; U.S. Pat. Nos. 5,576,198 and 5,925,806; WO2005/0544478), as well as methods and compositions to importpolynucleotides and/or polypeptides into a plastid, includingtranslational fusion to a transit peptide (e.g., Comai et al. (1988) JBiol Chem 263:15104-15109).

The SuR polynucleotides and polypeptides provide a means for regulatingplastid gene expression via a chemical inducer that readily enters thecell. For example, using the T7 expression system for chloroplasts(McBride et al. (1994) Proc Natl Acad Sci USA 91:7301-7305) the SuRcould be used to control nuclear T7 polymerase expression.Alternatively, an SuR-regulated promoter could be integrated into theplastid genome and operably linked to the polynucleotide(s) of interestand the SuR expressed and imported from the nuclear genome, orintegrated into the plastid. In all cases, application of a sulfonylureacompound is used to efficiently regulate the polynucleotide(s) ofinterest.

Any type of cell and/or organism, prokaryotic or eukaryotic, can be usedwith the SuR methods and compositions. For example, any bacterial cellsystem can be transformed with the compositions. For example, methods ofE. coli, Agrobacterium and other bacterial cell transformation, plasmidpreparation and the use of phages are detailed, for example, in CurrentProtocols in Molecular Biology (Ausubel, et al., (eds.) (1994) a jointventure between Greene Publishing Associates, Inc. and John Wiley &Sons, Inc.).

The SuR systems can be used with any eukaryotic cell line, includingyeasts, protists, algae, insect cells, avian or mammalian cells. Forexample, many commercially and/or publicly available strains of S.cerevisiae are available, as are the plasmids used to transform thesecells. For example, strains are available from the American Type CultureCollection (ATCC, Manassas, Va.) and include the Yeast Genetic StockCenter inventory, which moved to the ATCC in 1998. Other yeast lines,such as S. pombe and P. pastoris, and the like are also available. Forexample, methods of yeast transformation, plasmid preparation, and thelike are detailed, for example, in Current Protocols in MolecularBiology (Ausubel et al. (eds.) (1994) a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., see Unit 13 inparticular). Transformation methods for yeast include spheroplasttransformation, electroporation, and lithium acetate methods. Aversatile, high efficiency transformation method for yeast is describedby Gietz and Woods ((2002) Methods Enzymol 350:87-96) using lithiumacetate, PEG 3500 and carrier DNA.

The SuRs can be used in mammalian cells, such as CHO, HeLa, BALB/c,fibroblasts, mouse embryonic stem cells and the like. Many commerciallyavailable competent cell lines and plasmids are well known and readilyavailable, for example from the ATCC (Manassas, Va.). Isolatedpolynucleotides for transformation and transformation of mammalian cellscan be done by any method known in the art. For example, methods ofmammalian and other eukaryotic cell transformation, plasmid preparation,and the use of viruses are detailed, for example, in Current Protocolsin Molecular Biology (Ausubel et al. (eds.) (1994) a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,see, Unit 9 in particular). For example, many methods are available,such as calcium phosphate transfection, electroporation, DEAE-dextrantransfection, liposome-mediated transfection, microinjection as well asviral techniques.

Any plant species can be used with the SuR methods and compositions,including, but not limited to, monocots and dicots. Examples of plantsinclude, but are not limited to, corn (Zea mays), Brassica spp. (e.g.,B. napus, B. rapa, B. juncea), castor, palm, alfalfa (Medicago sativa),rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor,Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), prosomillet (Panicum miliaceum), foxtail millet (Setaria italica), fingermillet (Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), Arabidopsis thaliana,oats (Avena spp.), barley (Hordeum spp.), leguminous plants such as guarbeans, locust bean, fenugreek, garden beans, cowpea, mungbean, favabean, lentils, and chickpea, vegetables, ornamentals, grasses andconifers. Vegetables include tomatoes (Lycopersicon esculentum), lettuce(e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans(Phaseolus limensis), peas (Pisium spp., Lathyrus spp.), and Cucumisspecies such as cucumber (C. sativus), cantaloupe (C. cantalupensis),and musk melon (C. melo). Ornamentals include azalea (Rhododendronspp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscusrosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils(Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthuscaryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.Conifers include pines, for example, loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata), Douglas fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis), Sitkaspruce (Picea glauca), redwood (Sequoia sempervirens), true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea) and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow cedar(Chamaecyparis nootkatensis).

The plant cells and/or tissue that have been transformed may be growninto plants using conventional methods (see, e.g., McCormick et al.(1986) Plant Cell Rep 5:81-84). These plants may then be grown andself-pollinated, backcrossed, and/or outcrossed, and the resultingprogeny having the desired characteristic identified. Two or moregenerations may be grown to ensure that the characteristic is stablymaintained and inherited and then seeds harvested. In this mannertransformed/transgenic seed having a DNA construct comprising apolynucleotide of interest and/or modified polynucleotide encoding anSuR stably incorporated into their genome are provided. A plant and/or aseed having stably incorporated the DNA construct can be furthercharacterized for expression, agronomics and copy number.

Sequence identity may be used to compare the primary structure of twopolynucleotides or polypeptide sequences, describe the primary structureof a first sequence relative to a second sequence, and/or describesequence relationships such as variants and homologues. Sequenceidentity measures the residues in the two sequences that are the samewhen aligned for maximum correspondence. Sequence relationships can beanalyzed using computer-implemented algorithms. The sequencerelationship between two or more polynucleotides or two or morepolypeptides can be determined by computing the best alignment of thesequences and scoring the matches and the gaps in the alignment, whichyields the percent sequence identity and the percent sequencesimilarity. Polynucleotide relationships can also be described based ona comparison of the polypeptides each encodes. Many programs andalgorithms for comparison and analysis of sequences are known. Unlessotherwise stated, sequence identity/similarity values provided hereinrefer to the value obtained using GAP Version 10 (GCG, Accelrys, SanDiego, Calif.) using the following parameters: % identity and %similarity for a nucleotide sequence using GAP Weight of 50 and LengthWeight of 3, and the nwsgapdna.cmp scoring matrix; % identity and %similarity for an amino acid sequence using GAP Weight of 8 and LengthWeight of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff(1992) Proc Natl Acad Sci USA 89:10915-10919). GAP uses the algorithm ofNeedleman and Wunsch (1970) J Mol Biol 48:443-453, to find the alignmentof two complete sequences that maximizes the number of matches andminimizes the number of gaps.

Alternatively, polynucleotides and/or polypeptides can be evaluatedusing other sequence tools. For example, polynucleotides and/orpolypeptides can be evaluated using a BLAST alignment tool. A localalignment gaps consists simply of a pair of sequence segments, one fromeach of the sequences being compared. A modification of Smith-Watermanor Sellers algorithms will find all segment pairs whose scores cannot beimproved by extension or trimming, called high-scoring segment pairs(HSPs). The results of the BLAST alignments include statistical measuresto indicate the likelihood that the BLAST score can be expected fromchance alone. The raw score, S, is calculated from the number of gapsand substitutions associated with each aligned sequence wherein highersimilarity scores indicate a more significant alignment. Substitutionscores are given by a look-up table (see PAM, BLOSUM). Gap scores aretypically calculated as the sum of G, the gap opening penalty and L, thegap extension penalty. For a gap of length n, the gap cost would beG+Ln. The choice of gap costs, G and L is empirical, but it is customaryto choose a high value for G (10-15) and a low value for L (1-2). Thebit score, S′, is derived from the raw alignment score S in which thestatistical properties of the scoring system used have been taken intoaccount. Bit scores are normalized with respect to the scoring system,therefore they can be used to compare alignment scores from differentsearches. The E-Value, or expected value, describes the likelihood thata sequence with a similar score will occur in the database by chance. Itis a prediction of the number of different alignments with scoresequivalent to or better than S that are expected to occur in a databasesearch by chance. The smaller the E-Value, the more significant thealignment. For example, an alignment having an E value of e⁻¹¹⁷ meansthat a sequence with a similar score is very unlikely to occur simply bychance. Additionally, the expected score for aligning a random pair ofamino acid is required to be negative, otherwise long alignments wouldtend to have high score independently of whether the segments alignedwere related. Additionally, the BLAST algorithm uses an appropriatesubstitution matrix, nucleotide or amino acid and for gapped alignmentsuses gap creation and extension penalties. For example, BLAST alignmentand comparison of polypeptide sequences are typically done using theBLOSUM62 matrix, a gap existence penalty of 11 and a gap extensionpenalty of 1. Unless otherwise stated, scores reported from BLASTanalyses were done using the BLOSUM62 matrix, a gap existence penalty of11 and a gap extension penalty of 1.

UniProt protein sequence database is a repository for functional andstructural protein data and provides a stable, comprehensive, fullyclassified, richly and accurately annotated protein sequenceknowledgebase, with extensive cross-references and querying interfacesfreely accessible to the scientific community. The UniProt site has atool, UniRef, that provides a cluster of proteins have 50%, 90% or 100%sequence identity to a protein sequence of interest from the database.For example, using TetR(B) (UniProt reference P04483) gives a cluster of18 proteins having 90% sequence identity to P04483:

RefID Protein Name Species Length P04483 TetR class B from transposon E.coli 207 Tn10 B1VCF0 TetR protein E. coli 208 A0ZSZ1 Tetracyclineresistant gene Photobacterium sp. 208 repressor TC21 A4LA82 Tetracyclinerepressor protein Edwardsiella tarda 208 A4V9K4 Tetracycline repressorSalmonella enterica 208 A8R6K3 Tetracycline repressor protein Salmonellaenterica 208 subsp. enterica serovar Choleraesuis Q573N4 Tetracyclinerepressor protein uncultured 208 bacterium Q7BQ37 TetR Shigella flexneri208 Q9S455 TetR Salmonella typhi 208 A4IUI5 Tetracycline repressorprotein Yersinia ruckeri 207 R, class B Q1A2K5 Tetracycline resistanceE. coli 207 repressor protein Q6MXH5 TetR class B from transposonSerratia marcescens 207 tn10 Q79VX4 TetR protein Salmonella 207typhimurium Q7AZW7 Tet repressor protein Pasteurella 207 aerogenesQ7AK84 Repressor of tet operon Plasmid R100 207 Q6QR72 Tetracyclinerepressor protein E. coli 208 Q93F26 Tet repressor Shigella flexneri 2a208 Q8L0M9 Putative tetracycline repressor Neisseria 205 proteinmeningitidis

These protein sequences can be used as sources for sequence diversityfor protein design and/or directed evolution of the ligand bindingdomain. Further, these protein sequences can be used as sources foroperator binding domains for chimeric repressor proteins, or for designand/or evolution of the operator binding domain.

The properties, domains, motifs and function of tetracycline repressorsare well known, as are standard techniques and assays to evaluate anyderived repressor comprising one or more amino acid substitutions. Thestructure of the class D TetR protein comprises 10 alpha helices withconnecting loops and turns. The 3 N-terminal helices form theDNA-binding HTH domain, which has an inverse orientation as compared toHTH motifs in other DNA-binding proteins. The core of the protein,formed by helices 5-10, comprises the dimerization interface domain, andfor each monomer comprises the binding pocket for ligand/effector anddivalent cation cofactor (Kisker et al. (1995) J Mol Biol 247:260-180;Orth et al. (2000) Nat Struct Biol 7:215-219). Any amino acid change maycomprise a non-conservative or conservative amino acid substitution.Conservative substitutions generally refer to exchanging one amino acidwith another having similar chemical and/or structural properties (see,e.g., Dayhoff et al. (1978) Atlas of Protein Sequence and Structure,Natl Biomed Res Found, Washington, D.C.). Different clustering of aminoacids by similarity have been developed depending on the propertyevaluated, such as acidic vs. basic, polar vs. non-polar, amphipathicand the like and be used when evaluating the possible effect of anysubstitution or combination of substitutions.

Numerous variants of TetR have been identified and/or derived andextensively studied. In the context of the tetracycline repressorsystem, the effects of various mutations, modifications and/orcombinations thereof have been used to extensively characterize and/ormodify the properties of tetracycline repressors, such as cofactorbinding, ligand binding constants, kinetics and dissociation constants,operator binding sequence constraints, cooperativity, binding constants,kinetics and dissociation constants and fusion protein activities andproperties. Variants include TetR variants with a reverse phenotype ofbinding the operator sequence in the presence of tetracycline or ananalog thereof, variants having altered operator binding properties,variants having altered operator sequence specificity and variantshaving altered ligand specificity and fusion proteins. See, for example,Isackson and Bertrand (1985) Proc Natl Acad Sci USA 82:6226-6230; Smithand Bertrand (1988) J Mol Biol 203:949-959; Altschmied et al. (1988)EMBO J 7:4011-4017; Wissmann et al. (1991) EMBO J 10:4145-4152;Baumeister et al. (1992) J Mol Biol 226:1257-1270; Baumeister et al.(1992) Proteins 14:168-177; Gossen and Bujard (1992) Proc Natl Acad SciUSA 89:5547-5551; Wasylewski et al. (1996) J Protein Chem 15:45-58;Berens et al. (1997) J Biol Chem 272:6936-6942; Baron et al. (1997) NuclAcids Res 25:2723-2729; HelbI and Hillen (1998) J Mol Biol 276:313-318;Urlinger et al. (2000) Proc Natl Acad Sci USA 97:7963-7968; Kamionka etal. (2004) Nucl Acids Res 32:842-847; Bertram et al. (2004) J MolMicrobiol Biotechnol 8:104-110; Scholz et al. (2003) J Mol Biol 329:217-227; and patent publication US 2003/0186281.

The three-dimensional structures of tetracycline repressors, andtetracycline repressor variants, coupled to ligand and/or co-factor(s),and bound to operator sequence are known (see, for example, Kisker etal. (1995) J Mol Biol 247:260-280; Orth et al. (1998) J Mol Biol279:439-447; Orth et al. (1999) Biochemistry 38:191-198; Orth et al.(2000) Nat Struct Biol 7:215-219; Luckner et al. (2007) J Mol Biol368:780-790) providing extremely well characterized structure(s),identification of domains and individual amino acids associated withvarious functions and binding properties, and predictive model(s) forthe potential effects of any amino acid substitution(s), as well as thepossible structural bases for the phenotype(s) of known tetracyclinerepressor mutants. One example of percent sequence identity observedwithin tetracycline repressor family members is shown below.

% polypeptide sequence identity between TetR family members A E B D HTetR Class (P03038) (P21337) (P04483) (P0ACT4) (P51561) A (P03038) 10044 51 48 50 E (P21337) 100 51 49 50 B (P04483) 100 64 64 D (P0ACT4) 10058 H (P51561) 100

EXAMPLES Example 1 Evolution of TetR for Recognition by SulfonylureaCompounds

A. Computational Modeling

The 3-D crystal structures of the class D tetracycline repressor(isolated from E. coli; TET-bound dimer, 1DU7 (Orth et al. (2000) NatStruct Biol 7:215-219); and DNA-bound dimer, 1QPI (Orth et al. (2000)Nat Struct Biol 7:215-219)), were used as the design scaffold forcomputational replacement of the tetracycline (TET) molecule by thethifensulfuron-methyl (Ts, Harmony®) molecule in the ligand bindingpocket. TET and sulfonylureas (SUs) are generally similar in size andhave aromatic ring-based structures with hydrogen bond donors andacceptors, potentially allowing SU binding to a mutated TetR. However,there are notable differences between the tetracycline family and SUfamily of molecules. TET is internally rigid and fairly flat, with onehighly-hydrogen-bonding face with hydroxyls and ketones, logP ˜−0.3.Sulfonylureas (SUs) are more highly flexible and aromatic, with a coresulfonyl-urea moiety typically connecting a substituted benzene,pyridine, or thiophene (as in the case of Harmony®) on one side with asubstituted pyrimidine or 1,3,5-triazine on the other side. Althoughhaving different functional groups, the logP of Harmony® is similar(˜0.02 at pH 7) to that of tet. A best-posed Harmony® molecule waspositioned by molecular modeling in the TetR binding pocket in silico(FIG. 1). Based on this model, seventeen amino acid residue positions(60, 64, 82, 86, 100, 104, 105, 113, 116, 134, 135, 138 and 139 frommonomer A and positions 147, 151, 174 and 177 from monomer B, usingTetR(B) numbering) were determined to be in sufficiently close proximityto a docked Harmony® as to be recruited into a binding surface.Computational side-chain optimization was employed to design sets ofamino acids at each of the 17 positions deemed to be most compatiblewith SU binding. This resulted in a library with (4, 5, 4, 4, 5, 3, 8,11, 10, 10, 8, 8, 7, 9, 6, 7 and 5) amino acids at the 17 positions, fora total designed library size of 4×10¹³. The choice of amino acids atthe library positions was dictated by steric and physicochemicalconsiderations to fit ligand docking into the ligand pocket.

The wild type class B TetR from Tn10 was chosen as the starting moleculefor generation of shuffling derivatives (SEQ ID NO: 2). It is slightlydifferent than the sequence used in computational design (P0ACT4, classD, for which the high-resolution crystal structure 1DU7 is available),but only subtly affects ligand binding. A comparison of TetR(D) (SEQ IDNO: 401) and TetR(B) (SEQ ID NO: 2) is shown below with positionsinvolved in tet recognition and binding in bold:

  1        .         .         .         .         .         . 1DU7    SRLNRESVIDAALELLNETGIDGLTTRKLAQKLGIEQPTLYWHVKNKRALLDALAVEI Class B  MGSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEM 61        .         .         .         .         .         . 1DU7  LARHHDYSLPAAGESWQSFLRNNAMSFRRALLRYRDGAKVHLGTRPDEKQYDTVETQLRF Class B  LDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAF121        .         .         .         .         .         . 1DU7  MTENGFSLRDGLYAISAVSHFTLGAVLEQQEHTAALTDRPAAPDENLPPLLREALQIMDS Class B  LCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDH181       .          .        208 1DU7   DDGEQAFLHGLESLIRGFEVQLTALLQIVClass B   QGAEPAFLFGLELIICGLEKQLKCESGS-

The starting polynucleotide used to express TetR was synthesizedcommercially and restriction sites were added for functionality inlibrary construction and further manipulation (DNA2.0, Menlo Park,Calif., USA). Added restriction sites include an NcoI site at the 5′end, a SacI site 5′ of the ligand binding domain (LBD) and an AscI sitefollowing the stop codon. This allows library construction to belocalized in a ˜480 bp DNA segment containing the ligand binding regionto avoid inadvertent mutations in the other regions, such as the DNAbinding domain. The synthetic gene was operably linked downstream of anarabinose inducible promoter, P_(BAD), using Ncoi/AscI to create TetRexpression vector pVER7314 (FIG. 2). The addition of the NcoI site atthe 5′ end of the coding region resulted in the insertion of a glycineafter the N-terminal methionine at amino acid position one (SEQ ID NO:2). This sequence was used as the wild type TetR control in all assaysunless otherwise noted, and observed activity was equivalent to TetRwithout the serine insertion (SEQ ID NO: 1). However, all references toamino acid positions and changes designed and observed use the aminoacid numbering of wild type TetR(B) (207 aa) e.g., SEQ ID NO: 1.

B. Library Design

Due to the large number of designed substitutions at many positions inclose proximity with one another the computed library (Table 1, DesignedLibrary) was not easily encodable with a small number of degeneratecodons. This is particularly evident in sequence regions such as aminoacids 134, 135, 138 and 139, which could reasonably be encoded by asingle primer. For this reason, the sequence library fabricated andtested in the lab featured the designed amino acid set at 6/17positions, slightly enlarged at 1/17 positions, and fully degenerate(NNK codon) at 10/17 positions (Table 1). This resulted in much higherpredicted sequence diversity, a total of 3×10¹⁹ sequences.

TABLE 1 WT Resi- resi- Designed Actual due due Library Library  60 LA L K M A L K M  64 H A N Q H L A N Q H L  82 N A N S T A N S T  86 FM F W Y M F W Y 100 H H M F W Y All 20 aa's 104 R A R G A R G 105 PA N D G P S T V All 20 aa's 113 L A R N D Q E K M A R N D Q E K M S T VS T V I P L G H 116 Q A R N Q E I K M T V All 20 aa's 134 LA R I L K M F W Y V All 20 aa's 135 S A R N Q H K S T A R N Q H K S T138 G A H K M F S Y W All 20 aa's 139 H A R Q H L K Y All 20 aa's 147 EA R Q E H L K M Y All 20 aa's 151 H A Q H K I L All 20 aa's 174 IA R Q E L K M All 20 aa's 177 F A R L K M All 20 aa's

The constructed library, termed ‘L1’, was encoded with a total of fiftyoligonucleotides (Table 2) rather than the thousands that would havebeen required to completely specify the designed target library. Table 2also includes two PCR amplification primers.

TABLE 2 Oligo ID Oligo Sequence SEQ ID L1:01TATTGGCATGTAAAAAATAAGCGAGCTCTGCTCGACGCCTTA 833 L1:02GCCATTGAGATGAWGGATAGGCACCWGACTCACTTTGCCCT 834 L1:03GCCATTGAGATGAWGGATAGGCACMATACTCACTTTGCCCT 835 L1:04GCCATTGAGATGAWGGATAGGCACGCGACTCACTTTGCCCT 836 L1:05GCCATTGAGATGACGGATAGGCACCWGACTCACTTTGCCCT 837 L1:06GCCATTGAGATGACGGATAGGCACMATACTCACTTTGCCCT 838 L1:07GCCATTGAGATGACGGATAGGCACGCGACTCACTTTGCCCT 839 L1:08GCCATTGAGATGATGGATAGGCACCWGACTCACTTTGCCCT 840 L1:09GCCATTGAGATGATGGATAGGCACMATACTCACTTTGCCCT 841 L1:10GCCATTGAGATGATGGATAGGCACGCGACTCACTTTGCCCT 842 L1:11TTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCT 843 L1:12TTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATDCTGCT 844 L1:13AAAAGTTWTAGATGTGCTTTACTAAGTCATCGCGATGGAGCA 845 L1:14AAAAGTTGGAGATGTGCTTTACTAAGTCATCGCGATGGAGCA 846 L1:15AAAAGTATGAGATGTGCTTTACTAAGTCATCGCGATGGAGCA 847 L1:16AAAGTANNKTTAGGTACAGCGNNKACAGAAAAACAGTATGAA 848 L1:17AAAGTANNKTTAGGTACASGCNNKACAGAAAAACAGTATGAA 849 L1:18ACTVNSGAAAATNNKTTAGCCTTTTTATGCCAACAAGGTTTT 850 L1:19TCACTAGAGAATGCATTATATGCANNSRCCGCTGTGNNKNNK 851 L1:20TCACTAGAGAATGCATTATATGCANNSMGCGCTGTGNNKNNK 852 L1:21TCACTAGAGAATGCATTATATGCANNSMAKGCTGTGNNKNNK 853 L1:22TTTACTTTAGGTTGCGTATTGNNKGATCAAGAGNNKCAAGTC 854 L1:23GCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCG 855 L1:24CCATTATTACGACAAGCTNNKGAATTANNKGATCACCAAGGT 856 L1:25GCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGC 857 L1:26GGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCTTAAGGC 858 L1:27CCTATCCWTCATCTCAATGGCTAAGGCGTCGAGCAGAGCTCG 859 L1:28CCTATCCGCCATCTCAATGGCTAAGGCGTCGAGCAGAGCTCG 860 L1:29CCTATCCAGCATCTCAATGGCTAAGGCGTCGAGCAGAGCTCG 861 L1:30TTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTCWGGTG 862 L1:31TTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTATKGTG 863 L1:32TTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTCGCGTG 864 L1:33TAAAGCACATCTAWAACTTTTAGCGTTATTACGTAAAAAATC 865 L1:34TAAAGCACATCTCCAACTTTTAGCGTTATTACGTAAAAAATC 866 L1:35TAAAGCACATCTCATACTTTTAGCGTTATTACGTAAAAAATC 867 L1:36TAAAGCACATCTAWAACTTTTAGCAGHATTACGTAAAAAATC 868 L1:37TAAAGCACATCTCCAACTTTTAGCAGHATTACGTAAAAAATC 869 L1:38TAAAGCACATCTCATACTTTTAGCAGHATTACGTAAAAAATC 870 L1:39CGCTGTACCTAAMNNTACTTTTGCTCCATCGCGATGACTTAG 871 L1:40GCSTGTACCTAAMNNTACTTTTGCTCCATCGCGATGACTTAG 872 L1:41GGCTAAMNNATTTTCSNBAGTTTCATACTGTTTTTCTGTMNN 873 L1:42ATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAA 874 L1:43CAATACGCAACCTAAAGTAAAMNNMNNCACAGCGGYSNNTGC 875 L1:44CAATACGCAACCTAAAGTAAAMNNMNNCACAGCGCKSNNTGC 876 L1:45CAATACGCAACCTAAAGTAAAMNNMNNCACAGCMTKSNNTGC 877 L1:46TGTTTCCCTTTCTTCTTTAGCGACTTGMNNCTCTTGATCMNN 878 L1:47MNNAGCTTGTCGTAATAATGGCGGCATACTATCAGTAGTAGG 879 L1:48GAATAAGAAGGCTGGCTCTGCACCTTGGTGATCMNNTAATTC 880 L1:49TTTAAGTTGTTTTTCTAATCCGCATATGATCAATTCAAGGCC 881 L1:50GGGAACTTCGGCGCGCCTTAAGACCCACTTTCACA 882 L1:5′ CATGTAAAAAATAAGCGAGCTCTG883 L1:3′ GGGAACTTCGGCGCGCCTTAAGAC 884

Assembly of the ‘L1’ oligos was carried out by overlap extension (Ness,et al., (2002) Nat Biotech 20:1251-1255) to generate a PCR fragmentbordered by SacI/AscI restriction sites. Conditions for assembly of alllibrary fragments were as follows: oligonucleotides representing thelibrary are normalized to a concentration of 10 μM and then equalvolumes mixed to create a 10 μM pool. PCR amplification of libraryfragments was performed in six identical 25 μl reactions containing: 1 Mpooled library oligos; 0.5 μM of each rescue primer: L1:5′ and L1:3′ and200 M dNTP's in a Herculase II directed reaction (Stratagene, La Jolla,Calif., USA). Conditions for PCR were 98° C. for 1 min (initialdenature), followed by 25 cycles of 95° C. denature for 20 seconds,annealing for 45 seconds between 45° C. and 55° C. (gradient), thenextending the template for 30 seconds at 72° C. A final extension of 72°C. for 5 minutes completes the reaction. Wild type TetR(B) is excisedfrom the P_(BAD)-tetR expression vector pVER7314 by digestion withSacI/AscI. The pVER7314 backbone fragment is treated with calfintestinal phosphatase and purified, then the fully extended libraryfragment pool (˜500 bp) digested with SacI/AscI restriction enzymes areinserted to generate the L1 plasmid library. Approximately 50 randomclones from library L1 were sequenced and the information compiled forquality control purposes. The results indicated that nearly all aminoacids targeted in the diversity set were represented (data not shown).Sequencing revealed that 17% of the sequences contained stop codons.This is less than the predicted 27% (e.g., 10 positions having 1/32codons be a stop codon, 1−(31/32)¹⁰˜27%). Additionally, sequenceanalysis showed that 13% of the clones had frame shifts due to mistakesin the overlap extension process. Thus, overall approximately 30% of thelibrary consisted of clones encoding truncated polypeptides.

C. Screen Set Up

In order to test the library for rare clones reacting tothifensulfuron-methyl (Ts) a sensitive E. coli based genetic screen wasdeveloped. The screen is a modification of an established assay system(Wissmann et al. (1991) Genetics 128:225-232). The screen consists oftwo parts: a repressor pre-screen followed by an induction screen. Forthis purpose an E. coli strain was developed having bothfunctionalities. For the repressor prescreen a genetic cascade wasdeveloped whereby an nptIII gene encoding kanamycin resistance is underthe control of a lac promoter. The lac promoter is repressed by the Lacrepressor encoded by lacI, whose expression is in turn controlled by thetet promoter (PtetR). The tet promoter is repressed by TetR which blocksLacI production and thus ultimately enables kanamycin resistance to beexpressed.

Since the tet regulon has bivalent promoters, one promoter for tetR andone promoter for tetA, the same strain was engineered with the E. colilacZ gene encoding enzyme reporter β-galactosidase under control of thetetA promoter (PtetA). The dual regulon encoding both lacI and lacZ wasthen bordered by strong transcriptional terminators: the E. coli RNAribosomal operon terminator rrnB T1-T2 (Ghosh et al. (1991) J Mol Biol222:59-66) and the E. coli RNA polymerase subunit C terminator rpoC,such that spurious transcripts read in the direction of either tetpromoter would not interfere with expression of any other transcript. Inthe presence of functional TetR, the strain exhibits a lac⁻ phenotypeand colonies can be easily scored for induction by novel chemistry withX-gal, wherein induction gives increased blue colony color. In addition,induction with novel chemistry in liquid cultures can be measuredquantitatively by employing β-galactosidase enzyme assays with eithercolorimetric or fluorimetric substrates.

A further refinement of the host strain is that the to/C locus wasknocked out with the incoming Plac-nptIII reporter. This was done toobtain better penetration of SU compounds into E. coli (RobertLaRossa—DuPont: personal communication). A strong transcriptionalterminator, T22 from S. typhimurium phage P22, was placed upstream ofthe lac promoter to prevent unregulated leaky expression of theconditional kanamycin resistance marker. The name of the finalengineered strain is E. coli KM3.

The population of shuffled tetR LBD's was cloned into an Ap^(r)/ColE1based vector pVER7314 behind the P_(BAD) promoter. This was designed toenable fine control of TetR expression by variation of arabinoseconcentrations in the growth medium (Guzman et al. (1995) J Bacteriol177:4121-4130). Despite being under the control of the P_(BAD) promoter,TetR protein is expressed at a sufficient level in the absence of addedarabinose to enable selection for kanamycin resistance in strain KM3.Nevertheless, expression can be increased by addition of arabinose, forexample, if a change in assay stringency is desired.

D. Library Screening

Following assembly of L1 oligos and capture in vector pVER7314, theresulting library was transformed into E. coli strain KM3 and plated onLB containing 50 μg/ml carbenicillin to select for library plasmids, and60 μg/ml kanamycin to select for the active repressor population in theabsence of target ligand (“apo-repressors”). DNA sequence analysis ofthis selected population indicated that this step highly enrichedseveral library positions, suggesting that few amino acid combinationsin the ligand binding domain lead to a conformation compatible with DNAbinding by the N-terminal domain. In addition, this step eliminatedclones with premature stop codons and or frame shift mutations.Subsequently, these apo-repressor sequences were screened for alterationin repressor activity in the presence of Harmony® (Ts). This was done byreplica plating the Km^(r) pre-selected population from liquid culturesin 384-well format onto M9 agar containing 0.1% glycerol as carbonsource, 0.04% casamino acids (to prevent branched chain amino acidstarvation caused by sulfonylurea application), 50 μg/ml carbenicillinfor plasmid maintenance, 0.004% X-gal to detect-galactosidase activity,and +/−SU inducer Ts at 20 μg/ml. Initial hits were identified from apopulation of nearly 20,000 colonies screened for response to Tsfollowing incubation at 30° C. for 2 days. Fourteen putative ‘hits’identified were then re-tested under the same conditions but in 96-wellformat (FIG. 3).

DNA sequence analysis revealed that clones L1-3 and L1-19 are identicaland that the most intensely responding hits (L-2, -3(19), -5, -9, -11and -20) had significant enrichment at several library positions,indicating an involvement in ligand interaction, directly or indirectly.The same library was then re-screened to identify a further 10 hits tobring the total number of clones to 23.

All 23 putative hits were subsequently screened in the same plate assayformat with a panel of nine sulfonylurea (SU) compounds registered forcommercial use (Table 3), wherein 11 hits were found to respondsignificantly to other SU ligands (Table 4). For this experiment, E.coli clones encoding L1 hits or wt TetR (SEQ ID NO: 2) were arrayed in96-well format and stamped onto M9 X-gal assay media with or withouttest SU compounds at 20 μg/ml. Following 48 hrs growth at 30° C. theplates were digitally imaged and the colony color intensity converted torelative values of β-galactosidase activity. Inducers used:thifensulfuron (Ts), metsulfuron (Ms), sulfometuron (Sm),ethametsulfuron (Es), tribenuron (Tb), chlorimuron (Ci), nicosulfuron(Ns), rimsulfuron (Rs), chlorsulfuron (Cs) at 20 ppm andanhydrotetracycline (atc) as the positive control at 0.4 μM forinduction of wt TetR. Surprisingly, some sulfonylurea compounds,particularly chlorimuron, ethametsulfuron, and chlorsulfuron were morepotent activators than the starting ligand Harmony®.

TABLE 3 SU Compound Product Common Name Name Commercial UseThifensulfuron-methyl (Ts) Harmony ® Cereals, corn, soybeanMetsulfuron-methyl (Ms) Ally ® Cereals, pasture Sulfometuron-methyl (Sm)Oust ® Vegetation management Ethametsulfuron-methyl (Es) Muster ® CanolaTribenuron-methyl (Tb) Express ® Cereal, sunflower Chlorimuron-ethyl(Ci) Classic ® Soybean Nicosulfuron (Ns) Accent ® Corn Rimsulfuron (Rs)Matrix ® Corn, tomato, potato Chlorsulfuron (Cs) Glean ® Cereals

TABLE 4 Inducer clone None Ts Ms Sm Es Tb Ci Ns Rs Cs atc L1-2 1.0 1.61.9 4.7 5.8 1.7 13.6 1.3 1.3 4.1 1.2 L1-7 0.0 0.1 0.2 6.4 0.1 0.2 16.50.1 0.2 3.1 0.0 L1-9 0.3 1.1 1.2 0.6 11.8 0.4 9.8 0.3 0.4 23.6 0.3 L1-201.4 2.6 12.4 6.0 15.0 2.6 13.5 1.6 2.0 22.0 2.0 L1-22 0.1 0.0 0.1 17.20.3 0.3 10.4 0.2 0.1 0.2 0.0 L1-24 0.1 0.3 0.4 3.1 0.2 1.6 22.1 0.3 0.33.3 0.1 L1-28 0.0 0.1 18.8 1.1 0.8 0.3 14.6 0.1 0.2 5.8 0.0 L1-29 0.00.0 13.5 2.7 1.7 0.3 20.9 0.1 0.1 15.8 0.0 L1-31 0.3 0.9 0.5 0.9 13.70.1 1.1 0.5 0.4 1.4 0.4 L1-38 9.5 16.7 14.7 18.3 14.8 15.8 15.3 8.7 9.514.0 6.4 L1-44 0.2 1.9 2.9 0.4 2.4 0.4 6.7 0.4 0.3 12.0 0.2 TetR 0.0 0.00.0 0.1 0.0 0.1 0.1 0.1 0.1 0.0 25.0

The amino acid substitutions for these eleven top hits are summarized inTable 5. The sequences are compared to wild type TetR(B) and onlypositions having differences are shown using the numbering according toTetR(B) (e.g., SEQ ID NO: 1). A dash in the alignment indicates nochange relative to wt TetR ligand binding domain.

TABLE 5 60 64 82 86 100 104 105 113 116 134 135 138 139 147 151 164 174177 203 205 TetR(B) L H N F H R P L Q L S G H E H D I F C S L1-02 — A —— C A V M I R R I A W Y — R I — — L1-07 — N — W V A I H P I A R R V R —S Q — R L1-09 — A — M C G F A S M Q C I L L — L K — — L1-20 M Q — M F AW V L — N A T W K — H G S — L1-22 M — T Y C A I K N R Q R V F M — S L S— L1-24 — N S M L A V T S I R R T V R — K L — — L1-28 — A — M W A W P VS R V T T K — W L — — L1-29 M Q T M W — W P M W — C N S R — W S — —L1-31 — A — M M — A V R V R H W I M — Y Y — — L1-38 A — — M W A W T M WR T M R W — L G — — L1-44 — A — Y Y A V A — V K A G W S A V A — —

The initial screenings of library 1 also detected library members havingreverse repressor activity (SEQ ID NO: 1206-1213), wherein thepolypeptide was bound to the operator in the presence of SU ligand.These hits showed β-galactosidase expression without SU ligand, whichwas substantially reduced upon addition of the ligand, for examplethifensulfuron. These hits were subsequently screened in the same plateassay format as described above with the panel of nine sulfonylurea (SU)compounds registered for commercial use (Table 3), wherein 8 hits werefound to respond significantly to other SU ligands (Table 6).

TABLE 6 Inducer clone Blank Ts Ms Sm Es Tb Ci Ns Rs Cs atc L1-18 1.341.13 0.79 0.94 0.37 1.65 0.36 1.44 2.55 1.22 2.35 L1-21 2.88 0.79 0.892.39 0.61 2.13 0.07 2.74 2.31 0.89 2.81 L1-25 1.17 0.64 0.32 0.63 0.131.72 0.11 1.21 1.08 0.28 1.22 L1-33 7.59 5.51 4.29 5.02 2.11 4.71 0.765.34 10.32 3.74 8.25 L1-34 2.37 2.97 1.47 2.00 1.33 2.26 0.43 2.91 2.300.85 3.68 L1-36 1.52 0.48 0.38 0.50 0.20 0.57 0.21 1.81 1.84 0.24 1.70L1-39 3.65 1.42 0.75 0.91 0.60 0.97 0.49 3.03 4.72 0.89 4.92 L1-41 4.051.46 0.56 0.67 0.18 1.41 0.39 2.75 4.05 0.61 4.21 TetR 0.00 0.08 0.080.23 0.06 0.13 0.18 0.18 0.20 0.15 10.45

The amino acid substitutions for these eight reverse repressor hits (SEQID NO: 1206-1213 encoded by SEQ ID NO: 1214-1221) are summarized inTable 7. The sequences are compared to wild type TetR(B) and onlypositions having differences are shown using the numbering according toTetR(B) (e.g., SEQ ID NO: 1). A dash in the alignment indicates nochange relative to wt TetR ligand binding domain.

TABLE 7 Clone 60 63 64 82 84 86 100 104 105 113 116 121 134 135 138 139147 151 163 174 177 205 206 TetR wt L H H N K F H R P L Q C L S G H E HT I F S G SU-TetR-18 — — L — — M W G F K R — I R S R Q P — V — — ESU-TetR-21 — — A — — — C A G — C — R — V C F M — A L — — SU-TetR-25 — —A T — M L A T — L Y W Q W R I T — V K T — SU-TetR-33 — — A N — M Q A A —K — H — T Q R G — R R — — SU-TetR-34 A — N A R M Y A R T V — V R P R L R— R T — — SU-TetR-36 A — — A — M R A W H V — — K S G K M — T V — —SU-TetR-39 M — Q T — Y I — W R V — W A — P R R — L M — — SU-TetR-41 — NQ — — W M — N A G — C R W T D T S M K — —E. Correlation of First Round Shuffling Results with the StructuralModel

Significant enrichment occurred at most library positions, whereenrichment includes biases favoring particular amino acids and biasesdisfavoring particular amino acids. The initial screening involved twostages to identify both repressor and de-repressor functions. Enrichmentoccurred in both stages of screening. In the first stage, positions wereenriched by the selection for “apo repressors”, that is, proteins thatrepress gene transcription in the absence of ligand. In the secondstage, positions were enriched by the selection for “activators”, thatis, proteins that allow gene transcription in the presence of ligand.Positions may be enriched by either selection criterion, by bothcriteria, or by neither. The first-round screening results for repressoractivity are summarized below:

Amino Acid Bias Observed Position Apo repressor SU Induction Both 60 L(not K) 64 Q, N (not L, A) 82 N (not A, T) A (not N, S) 86 (not M) M(not W) 100 R (not K, Q) C, W (not H, K, Q) 104 G A 105 C, G, L, V (notH, K) L, W (not G, S) L 113 A (not G, P) A, I (not D, G) A 116 (not GL)M, V (not A, R) 134 M, S I, R, W (not G) 135 K, R (not H, S) Q, R (notA, T) R 138 (not T) A, C, R, V (not L, P, Q, T) 139 R (not H) T (not L,P) 147 (not A, C) R, W (not A, S) 151 R (not C, G, Q) M, R (not V) R 174V (not L, R) W (not F, L) 177 T (not S) K, L (not P, T)

Enrichment at the activator level was consistent with the computationalmodel of SU binding: sterically-selected positions (e.g., 60, 86, 104,105, 113, 138, 139) occurred in closest proximity to the modeled ligand,electrostatically-selected positions (e.g., 135, 147, 151, 177) occurrednear the modeled sulfonyl moiety, and aromatically-selected positions(e.g., 100, 105, 147, 174) were modeled to form aromatic stackinginteractions with the planar ring structures in thifensulfuron.Enrichment at the apo repressor level was consistent with the predictedmode of DNA binding: enriched positions were modeled to be capable ofmodulating association of the repressor protein with the DNA operatorsequence.

In the case of the SU induction screen, enrichment was evidenced by bothover-represented amino acids that were modeled to form favorableinteractions with the ligand (e.g., methionine (M) and valine (V) atposition 116 were modeled to pack well against the triazine ring of theSU molecule) and by under-represented amino acids that were modeled toproduce unfavorable interactions with the ligand (e.g., tryptophan (‘W’)at position 86 was modeled to be too large to accommodate ligand inbinding pocket). In the case of the apo repressor, enrichment took theform both of over-represented amino acids that were modeled to give riseto a functional repressor conformation capable of binding the DNAoperator sequence in the absence of ligand (e.g., alanine (‘A’) atposition 113, which maintains the structural integrity of this α-helix)and of under-represented amino acids that were modeled to disrupt theactively repressing conformation in the absence of ligand (e.g., glycineand proline (‘P’) at position 113, which reduce the structural integrityof this α-helix).

Results from different rounds of screening and selection may producealtered enrichments at some positions, as the result of interactionswith other amino acids selected, or with the small molecule. Enrichedsequences only demonstrate that side-chains can contribute to activeinducers, and do not rule out any amino acids. Thus, selected hits arelikely to represent only a subset of possible active sequences. Avariety of possible ligand-binding modes and protein interactions may bepossible for the same SU molecule, and thus enrichment of several typesof side-chains at a specific position may represent multiple populationsof active proteins. Computational modeling of the enriched sequences isuseful insofar as it enables the prioritization of amino acid diversityfor rounds of screening and selection.

Altogether, these enrichment results support the overall computationalmodel and facilitated additional design. Several positions which wereconstructed as fully-degenerate codons (all 20 amino acids) returnedfirst-round screening results consistent with the suggestedcomputational model. For example, computational modeling suggested thatthe aromatic side-chains W, Y and F at position 100 would favor SUbinding. The first-round library was screened with a degenerate codon atthis position, and the amino acids W, Y and F were significantlyenriched. Designed libraries allow sequence diversity to be narrowed atlibrary positions, with an emphasis on decreasing diversity at coupledpositions such that fully degenerate codons may be avoided.Additionally, more positions could be recruited for diversification toachieve greater coverage of a higher-quality, more focused sequencelibrary. This allows construction of a library with sufficiently fewmembers to screen with good coverage, yet sufficient diversity todiscover sequences with detectable activity. Such sequences may then beimproved by introducing more diversity at library positions, with ascreen or selection choosing optimal clones, independent of modelpredictions. In this way, the combination of computational modeling anddirected evolution allows the generation of engineered sequencesunlikely to be discovered by either technique separately.

F. Second-round Shuffling

The original library was designed to thifensulfuron, but once inductionactivity was established with other SU compounds having potentiallybetter soil and in planta stability properties than the original ligand,the evolution process was re-directed towards these alternative ligands.Of particular interest were herbicides metsulfuron, sulfometuron,ethametsulfuron and chlorsulfuron. For this objective, parental clonesL1-9, -22, -29 and -44 were chosen for further shuffling. Clone L1-9 hasstrong activity on both ethametsulfuron and chlorsulfuron; clone L1-22has strong sulfometuron activity; clone L1-29 has moderate metsulfuronactivity; and clone L1-44 has moderate activity on metsulfuron,ethametsulfuron and chlorsulfuron (Table 4). No clones found in theinitial screen were exceptionally reactive to metsulfuron. These fourclones were also chosen due to their relatively strong repressoractivity, showing low β-gal background activity without inducer. Strongrepressor activity is important for establishing a system which is bothhighly sensitive to the presence od inducer, and tightly off in theabsence of inducer.

Based on the sequence information from parental clones L1-9, -22, -29and -44, two second round libraries were designed, constructed andscreened. The first library, L2, consisted of a ‘family’ shuffle wherebythe amino acid diversity between the selected parental clones was variedusing synthetic assembly of oligonucleotides to find clones improved inresponsiveness to any of the four new target ligands. A summary of thediversity used and the resulting hit sequences for library L2 is shownin Table 8.

TABLE 8 Amino acid residue position Clone 60 64 82 86 100 104 105 113116 134 wt L H N F H R P L Q L Parents L1-9 — A — M C G F A S M L1-22 M— T Y C A I K N R L1-29 M Q T M W — W P M W L1-44 — A — Y Y A V A — VHits L2-2 — Q — M C — F K — V L2-9 M Q — M Y — W A — W L2-10 — A — M W GW K M M L2-13 — Q — M C — W A — W L2-14 M A — M C — W A M V L2-18 M Q TM W — W A — M L1-45 A Q — W W G L P V T Unselected random random randomrandom W > R >> W > V > random random random frequency C, Y G, A I, FInducer Clone 135 138 139 147 151 164 174 177 203 preference wt S G H EH D I F C atc Parents L1-9 Q C I L L — L K — 4, 9 (weak) L1-22 Q R V F M— S L S 3 L1-29 — C N S R — W S — 9 (weak) L1-44 K A G W S A V A —9 (weak) Hits L2-2 — R I W M — W L — 4 (inverse) L2-9 — A I W S — S K —9 (leaky) L2-10 — R I L L — W K — 4 (leaky) L2-13 Q R I S M — V K — 9L2-14 — R V F S A L K — 9 L2-18 — R N F L A W K — 9 L1-45 Q R — G R — AL — 3, 4 Unselected S >> A >> H, N > random random random random randomC >> S frequency Q, K C, R V > I

The oligonucleotides used to construct the library are shown in Table 9.The L2 oligonucleotides were assembled, cloned and screened as per theprotocol described for library L1 except that each ligand was tested at2 ppm to increase the stringency of the assay, which is a 10-foldreduction from 1st round library screening concentration.

TABLE 9 Oligo Sequence SEQ ID L2:01TATTGGCATGTAAAAAATAAGCGAGCTCTGCTCGACGCCTTA 885 L2:02GCCATTGAGATGWTGGATAGGCACCASACTCACTTTTGCCCT 886 L2:03GCCATTGAGATGWTGGATAGGCACGCAACTCACTTTTGCCCT 887 L2:04TTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAMTGCT 888 L2:05AAAAGTTACAGATGTGCTTTACTAAGTCATCGCGATGGAGCA 889 L2:06AAAAGTATGAGATGTGCTTTACTAAGTCATCGCGATGGAGCA 890 L2:07AAAGTATRTTTAGGTACACGCDTCACAGAAAAACAGTATGAA 891 L2:08AAAGTATRTTTAGGTACACGCTGGACAGAAAAACAGTATGAA 892 L2:09AAAGTATRTTTAGGTACAGSTDTCACAGAAAAACAGTATGAA 893 L2:10AAAGTATRTTTAGGTACAGSTTGGACAGAAAAACAGTATGAA 894 L2:11AAAGTATGGTTAGGTACACGCDTCACAGAAAAACAGTATGAA 895 L2:12AAAGTATGGTTAGGTACACGCTGGACAGAAAAACAGTATGAA 896 L2:13AAAGTATGGTTAGGTACAGSTDTCACAGAAAAACAGTATGAA 897 L2:14AAAGTATGGTTAGGTACAGSTTGGACAGAAAAACAGTATGAA 898 L2:15ACTAAAGAAAATARCTTAGCCTTTTTATGCCAACAAGGTTTT 899 L2:16ACTAAAGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTT 900 L2:17ACTAAAGAAAATATGTTAGCCTTTTTATGCCAACAAGGTTTT 901 L2:18ACTSCTGAAAATARCTTAGCCTTTTTATGCCAACAAGGTTTT 902 L2:19ACTSCTGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTT 903 L2:20ACTSCTGAAAATATGTTAGCCTTTTTATGCCAACAAGGTTTT 904 L2:21TCACTAGAGAATGCATTATATGCARTGAGTGCTGTGGCTAWT 905 L2:22TCACTAGAGAATGCATTATATGCARTGAGTGCTGTGGCTGKT 906 L2:23TCACTAGAGAATGCATTATATGCARTGAGTGCTGTGYGCAWT 907 L2:24TCACTAGAGAATGCATTATATGCARTGAGTGCTGTGYGCGKT 908 L2:25TCACTAGAGAATGCATTATATGCARTGMAAGCTGTGGCTAWT 909 L2:26TCACTAGAGAATGCATTATATGCARTGMAAGCTGTGGCTGKT 910 L2:27TCACTAGAGAATGCATTATATGCARTGMAAGCTGTGYGCAWT 911 L2:28TCACTAGAGAATGCATTATATGCARTGMAAGCTGTGYGCGKT 912 L2:29TCACTAGAGAATGCATTATATGCAWGGAGTGCTGTGGCTAWT 913 L2:30TCACTAGAGAATGCATTATATGCAWGGAGTGCTGTGGCTGKT 914 L2:31TCACTAGAGAATGCATTATATGCAWGGAGTGCTGTGYGCAWT 915 L2:32TCACTAGAGAATGCATTATATGCAWGGAGTGCTGTGYGCGKT 916 L2:33TCACTAGAGAATGCATTATATGCAWGGMAAGCTGTGGCTAWT 917 L2:34TCACTAGAGAATGCATTATATGCAWGGMAAGCTGTGGCTGKT 918 L2:35TCACTAGAGAATGCATTATATGCAWGGMAAGCTGTGYGCAWT 919 L2:36TCACTAGAGAATGCATTATATGCAWGGMAAGCTGTGYGCGKT 920 L2:37TTTACTTTAGGTTGCGTATTGTKGGATCAAGAGAGMCAAGTC 921 L2:38TTTACTTTAGGTTGCGTATTGTKGGATCAAGAGMTGCAAGTC 922 L2:39TTTACTTTAGGTTGCGTATTGTYTGATCAAGAGAGMCAAGTC 923 L2:40TTTACTTTAGGTTGCGTATTGTYTGATCAAGAGMTGCAAGTC 924 L2:41GCTAAAGAAGAAAGGGAAACACCTACTACTGMTAGTATGCCG 925 L2:42CCATTATTACGACAAGCTAGTGAATTATTGGATCACCAAGGT 926 L2:43CCATTATTACGACAAGCTAGTGAATTAKCAGATCACCAAGGT 927 L2:44CCATTATTACGACAAGCTAGTGAATTAAAGGATCACCAAGGT 928 L2:45CCATTATTACGACAAGCTTKGGAATTATTGGATCACCAAGGT 929 L2:46CCATTATTACGACAAGCTTKGGAATTAKCAGATCACCAAGGT 930 L2:47CCATTATTACGACAAGCTTKGGAATTAAAGGATCACCAAGGT 931 L2:48CCATTATTACGACAAGCTGTAGAATTATTGGATCACCAAGGT 932 L2:49CCATTATTACGACAAGCTGTAGAATTAKCAGATCACCAAGGT 933 L2:50CCATTATTACGACAAGCTGTAGAATTAAAGGATCACCAAGGT 934 L2:51GCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGC 935 L2:52GGATTAGAAAAACAACTTAAATSCGAAAGTGGGTCTTAA 936 L2:53CCTATCCAWCATCTCAATGGCTAAGGCGTCGAGCAGAGCTCG 937 L2:54TTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTSTGGTG 938 L2:55TTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTTGCGTG 939 L2:56TAAAGCACATCTGTAACTTTTAGCAKTATTACGTAAAAAATC 940 L2:57TAAAGCACATCTCATACTTTTAGCAKTATTACGTAAAAAATC 941 L2:58GCGTGTACCTAAAYATACTTTTGCTCCATCGCGATGACTTAG 942 L2:59ASCTGTACCTAAAYATACTTTTGCTCCATCGCGATGACTTAG 943 L2:60GCGTGTACCTAACCATACTTTTGCTCCATCGCGATGACTTAG 944 L2:61ASCTGTACCTAACCATACTTTTGCTCCATCGCGATGACTTAG 945 L2:62GGCTAAGYTATTTTCTTTAGTTTCATACTGTTTTTCTGTGAH 946 L2:63GGCTAATTGATTTTCTTTAGTTTCATACTGTTTTTCTGTGAH 947 L2:64GGCTAACATATTTTCTTTAGTTTCATACTGTTTTTCTGTGAH 948 L2:65GGCTAAGYTATTTTCAGSAGTTTCATACTGTTTTTCTGTGAH 949 L2:66GGCTAATTGATTTTCAGSAGTTTCATACTGTTTTTCTGTGAH 950 L2:67GGCTAACATATTTTCAGSAGTTTCATACTGTTTTTCTGTGAH 951 L2:68GGCTAAGYTATTTTCTTTAGTTTCATACTGTTTTTCTGTCCA 952 L2:69GGCTAATTGATTTTCTTTAGTTTCATACTGTTTTTCTGTCCA 953 L2:70GGCTAACATATTTTCTTTAGTTTCATACTGTTTTTCTGTCCA 954 L2:71GGCTAAGYTATTTTCAGSAGTTTCATACTGTTTTTCTGTCCA 955 L2:72GGCTAATTGATTTTCAGSAGTTTCATACTGTTTTTCTGTCCA 956 L2:73GGCTAACATATTTTCAGSAGTTTCATACTGTTTTTCTGTCCA 957 L2:74ATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAA 958 L2:75CAATACGCAACCTAAAGTAAAAWTAGCCACAGCACTCAYTGC 959 L2:76CAATACGCAACCTAAAGTAAAAMCAGCCACAGCACTCAYTGC 960 L2:77CAATACGCAACCTAAAGTAAAAWTGCRCACAGCACTCAYTGC 961 L2:78CAATACGCAACCTAAAGTAAAAMCGCRCACAGCACTCAYTGC 962 L2:79CAATACGCAACCTAAAGTAAAAWTAGCCACAGCTTKCAYTGC 963 L2:80CAATACGCAACCTAAAGTAAAAMCAGCCACAGCTTKCAYTGC 964 L2:81CAATACGCAACCTAAAGTAAAAWTGCRCACAGCTTKCAYTGC 965 L2:82CAATACGCAACCTAAAGTAAAAMCGCRCACAGCTTKCAYTGC 966 L2:83CAATACGCAACCTAAAGTAAAAWTAGCCACAGCACTCCWTGC 967 L2:84CAATACGCAACCTAAAGTAAAAMCAGCCACAGCACTCCWTGC 968 L2:85CAATACGCAACCTAAAGTAAAAWTGCRCACAGCACTCCWTGC 969 L2:86CAATACGCAACCTAAAGTAAAAMCGCRCACAGCACTCCWTGC 970 L2:87CAATACGCAACCTAAAGTAAAAWTAGCCACAGCTTKCCWTGC 971 L2:88CAATACGCAACCTAAAGTAAAAMCAGCCACAGCTTKCCWTGC 972 L2:89CAATACGCAACCTAAAGTAAAAWTGCRCACAGCTTKCCWTGC 973 L2:90CAATACGCAACCTAAAGTAAAAMCGCRCACAGCTTKCCWTGC 974 L2:91TGTTTCCCTTTCTTCTTTAGCGACTTGKCTCTCTTGATCCMA 975 L2:92TGTTTCCCTTTCTTCTTTAGCGACTTGCAKCTCTTGATCCMA 976 L2:93TGTTTCCCTTTCTTCTTTAGCGACTTGKCTCTCTTGATCARA 977 L2:94TGTTTCCCTTTCTTCTTTAGCGACTTGCAKCTCTTGATCARA 978 L2:95ACTAGCTTGTCGTAATAATGGCGGCATACTAKCAGTAGTAGG 979 L2:96CMAAGCTTGTCGTAATAATGGCGGCATACTAKCAGTAGTAGG 980 L2:97TACAGCTTGTCGTAATAATGGCGGCATACTAKCAGTAGTAGG 981 L2:98GAATAAGAAGGCTGGCTCTGCACCTTGGTGATCCAATAATTC 982 L2:99GAATAAGAAGGCTGGCTCTGCACCTTGGTGATCTGMTAATTC 983 L2:100GAATAAGAAGGCTGGCTCTGCACCTTGGTGATCCTTTAATTC 984 L2:101TTTAAGTTGTTTTTCTAATCCGCATATGATCAATTCAAGGCC 985 L2:102GGGAACTTCGGCGCGCCTTAAGACCCACTTTCGSA 986G. Screening Results for Library L2

Nearly 8,000 colonies arising from the repressor prescreen weresubjected to the activation screen on M9 assay medium containingpotential inducers ethametsulfuron, sulfometuron, metsulfuron orchlorsulfuron at 2 ppm. After 48 hours of incubation at 30° C. theplates were analyzed. Twelve putative hits from these plates werere-arrayed into 96-well format and retested on the same set of media(Table 10). Only clone L2-14 had a strong induction response and tightregulation to any of the inducers at this lower 2 ppm dose, wherein ithad a strong response to Cs and low background without inducer. CloneL2-18 had a moderate response to this ligand and low background. CloneL2-9 also responded well to Cs, but had higher background activitywithout inducer. No isolates showed a significant response tometsulfuron. An unexpected observation was that parent clone L1-9 hassensitivity to 2 ppm Es. Sequence analysis of the hit clones fromlibrary 2 (Table 6) indicates that F86M, G138R and F177K were preferredsubstitutions in the responding hits. This is especially striking atposition 138 where A is far over represented in the unselectedpopulation and yet five clones have R at this position while only onehas alanine. R105W could also be important, but in the random sequencepopulation W105 was already biased over C or Y.

TABLE 10 Inducer No Sample inducer Ms Sm Es Cs L1 parents L1-9 0.9 0.90.9 14.8 2.2 L1-22 0.2 0.2 1.8 0.2 0.2 L1-29 0.1 0.3 0.2 0.3 0.2 L1-440.6 0.5 0.6 1.0 1.9 L2 hits L2-9 2.2 4.2 6.3 5.0 14.0 L2-10 1.3 1.1 1.77.7 1.7 L2-13 1.8 1.9 2.4 2.0 8.4 L2-14 0.5 1.0 0.9 0.9 17.0 L2-18 0.30.1 0.2 0.3 4.6 TetR 0.1 0.0 0.1 0.1 0.1H. Second Round Library L4 Assembly

Another second round library, L4, was created from syntheticoligonucleotides using clone L1-9 as the template and injecting novelamino acid diversity into the sequence based on phylogenetic comparisonof 34 TetR and related molecules. A multiple sequence alignment of SEQID NO: 1 and SEQ ID NO: 402-433 generated using GCG SeqWeb PILEUP(Accelrys, San Diego, Calif.) under default parameters of gap weight=8,gap length weight=2, and the BLOSUM62 matrix is shown below:

1                                                   50 ZP_01558383~~~mkdtg.a rltrdtvmra aldllnevgi dglstrrlae rlgvqsptly YP_772551~~~mkdts.t rltrdtvmra aldllnevgi dglstrrlae rlgvqsptly YP_620166~~~mkdtg.t rltrdtvlra alnlldevgi dglstrrlae rlgvqsptly EAY62734miemkdtg.a rltrdtvlra alnlldevgi dglstrrlae rlgvqsptly YP_368094~~~mkdtg.a rltrdtvlra alelldevgi dglstrklae rlgvqsptly AAP93923~mseknta.a rltretvlrg alallddigi dalstrrlaq hlgvqsptly AAW66496~mskkdiapq rltreivlrt aldmlneegi dsittrklaq rlgiksptly CAA24908~~~~~~~~mt klqpntvira aldllnevgv dglttrklae rlgvqqpaly P03038~~~~~~~~mt klqpntvira aldllnevgv dglttrklae rlgvqqpaly ABS19067~~~~~~~~mi klqpntviry aldllnevgv ealttrklak rlgvqqpaly NP 387462~~~~~~~~mn klqreavirt alellndvgm eglttrrlae rlgvqqpaly NP 387455~~~~~~~~mk klqreavirt alellndvgm eglttrrlae rlgvqqpaly AAR96033~~~~~~~~mn klqreavirt alellndvgm eglttrrlae rlgvqqpaly NP 511232~~~~~~~~mn klqreavirt alellndvgm eglttrrlae rlgvqqpaly AAW83818~~~~~~~~mt klqpntvira aldllnevgv dglttrklae rlgvqqpaly AAD25094~~~~~~~~mt kldkgtviaa alellnevgm dslttrklae rlkvqqpaly AB014708~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~ly P51560~~~~~~~~mt kldkgtviaa glellnevgm dslttrklae rlkvqqpaly AAD25537~~~~~~~~mt kldkgtviaa alellnevgm dslttrklae rlrvqqpaly YP_001220607~~~~~~~~mt kldretviqa alellnevgv dnittrklae rlkvqqpaly YP_001370475~~~~mvsala klhrdaviqt alellnevge eglttrrlae rlgvqqpaly P21337~~~~~~~~ma rlslddvism altlldsegl eglttrklaq slkieqptly AAA98409~~~~~~~~ma rlslddvism altlldsegl eglttrklaq slkieqptly CAC81917~~~~~~~~ma rlslddvism altlldregl eglttrnvaq slkieqptly P51561~~~~~~~~ma kldkeqvidd alillnevgi eglttrnvaq kigveqptly ZP_00132379~~~~~~~~ma kldkeqvidn alillnevgi eglttrklaq kigveqptly AAD12754~~~~~~~~ma kldkeqvidn alillnevgm eglttrklaq klgveqptly P04483~~~~~~~~ms rldkskvins alellnevgi eglttrklaq klgveqptly A26948~~~~~~~~ms rldkskvins alellnevgi eglttrklaq klgveqptly CAC80726~~~~~~~mma rldkkrvies alalldevgm eglttrklaq klnieqpsly P0ACT4~~~~~~~~MA RLNRESVIDA ALELLNETGI DGLTTRKLAQ KLGIEQPTLY ZP_01567051~~~~~~~~ma kirrdeivda alalldeggl dalttrrlaq rlgvesaaly NP_824556~~~mvtqrsp kldkkqvvet alrllneagl dgltlraiak elnvqapaly51                                                 100 ZP_01558383whfrnkaell damaeaimle rhgaslprpg dawdawllen arsfrralla YP_772551whfrnkaell damaeaimle rhgaslprpg dtwdawllen argfrralla YP_620166whfrnkaell damaeaimle rhgeslprpg dvwdvwlaen arsfrralla EAY62734whfrnkaell damaeaimle rhgeslprpg dvwdvwlaen arsfrralla YP_368094whfrnkgell damaeaimle rhdaslprpg eawdawlldn arsfrralla AAP93923whfknkaell kamaetimld .hreevpadm p.wqawvtan ainfrralla AAW66496whfknksllm eamaetiine hhlvslpidg mtwqdwllan sysfrralla CAA24908whfrnkrall dalaeamlae nhstsvprad ddwrsfltgn arsfrqalla P03038whfrnkrall dalaeamlae nhthsvprad ddwrsflign arsfrqalla ABS19067whfrnkrall dalaeamlae nhthsvprvd ddwrsflign arsfrqalla NP_387462whfknkrall dalaeamlti nhthstprdd ddwrsflkgn acsfrralla NP_387455whfrnkrall dalaeamlti nhthstprde ddwrsflkgn acsfrralla AAR96033whfknkrall dalaeamlti nhthstprdd ddwrsflkgn acsfrralla NP_511232whfknkrall dalaeamlti nhthstprdd ddwrsflkgn acsfrralla AAW83818whfrnkrall dalaeamlae nhthsvprad ddwrsflkgn acsfrralla AAD25094whfqnkrall dalaeamlae rhtrslpeen edwrvflken alsfrtalls AB014708whfqnkrall dalaeamlae rhtrslpeen edwrvflken alsfrtalls P51560whfqnkrall dalpeamlre rhtrslpeen edwrvflken alsfrtalls AAD25537whfpskrall dalaeamlte rhtrslpeen edwrvflken alsfrkalls YP_001220607whfrnkrall dalseamlek nhtrtvpqtg edwrvflken alsfrsalls YP_001370475whfknkrvll dalaetilae hhdhalprag enwrhflien ahsfrrallt P21337whvrnkqtlm nmlseailak hhtrsaplpt eswqqflgen alsfrkallv AAA98409whlrnkqtlm nmlseailak hhtrsaplpt eswqqflgen alsfrkallv CAC81917whvrnkqtlm nmlseailak hhtrsvplpt eswqqflgen alsfrkallv P51561whvknkrall dalaetilqk hhhhvlplpn etwqdflrnn aksfrqallm ZP_00132379whvknkrall dalaetilqk hhhhvlplpn etwqdflrnn aksfrqallm AAD12754whvknkrall dalaetilqk hhhhvlplan eswqdflrnn aksfrqallm P04483whvknkrall dalaiemldr hhthfcpleg eswqdflrnn aksfrcalls A26948whvknkrall dalaiemldr hhthfcpleg eswqdflrnn aksfrcalls CAC80726whvknkrall dalsveillr hhdhfqpqkg eywadflren aksfrralls P0ACT4WHVKNKRALL DALAVEILAR HHDYSLPAAG ESWQSFLRNN AMSFRRALLR ZP_01567051whyrdksvll aemaavalar hhtldvpadt aqwdawfadn arsfrralla NP_824556whfknkqall dematemyrr mtegahlapg aswgerllhg nralrtallg101                                                150 ZP_01558383yrdgarlhag tr.prtlhfg sierkvalla eagfapdeav dvmyalgrfv YP_772551yrdgarlhag tr.prtlhfd sierkvalla dagfapdeav dvmyalgrfv YP_620166yrdgarlhag tr.pralhfs sierkvallg eagfkpdeav dvmvaigrfv EAY62734yrdgarlhag tr.pralhfs sierkvallg eagfkpdeav dvmvaigrfv YP_368094yrdgarlhag tr.pralhfs sierkvallg dagfapdeav dvmyalgrfv AAP93923yrdgarlhag tr.pgepgfa iieakvallc ragftpehav nllfavgrfv AAW66496yrdgarlhar ts.psqghfn tieaqvalls hagfspveav allmtlgrfi CAA24908yrdgarihag tr.pgapqme tadaqlrflc eagfsagdav nalmtisyft P03038yrdgarihag tr.pgapqme tadaqlrflc eagfsagdav nalmtisyft ABS19067yrdgarihag tr.pgapqme vvdaqlrflc eagfsawdav nalmtisyft NP_387462yrdgarihag tr.saapqme kadaqlrflc dagfsagdat yalmaisyft NP_387455yrdgarihag tr.paapqme kadaqlrflc dagflagdat yalmaisyft AAR96033yrdgarihag tr.paapqme kadaqlrflc dagfsagdat yalmaisyft NP_511232yrdgarihag tr.paapqme kadaqlrflc dagfsagdat yalmaisyft AAW83818yrdgarihag tr.paapqme kadaqlrflc dagfsagdat yalmaisyft AAD25094yrdgarihag tr.ptepnfg taetqirflc aegfcpkrav walrayshyv AB014708yrdgarihag tr.ptepnfg taetqirflc aegfcpkrav walrayshyv P51560yrdgarihag tr.ptepnfg taetqirflc aegfcpkrav walrayshyv AAD25537yrdgarihag tr.ptephyg taeaqirflc tagfspkrav walwayshyv YP_001220607yrdgarihag tr.ptsagye rvekqirflc esgfeqpdav ralvivshyt YP_001370475yrdgahihag tr.pnnnqag qaetqiefli qagftpanaa raliaishyv P21337hrdgarlhig ts.ptppqfe qaeaqlrcic dagfsveeal filqsishft AAA98409hrdgarlhig ts.ptppqfe qaeaqlrcic dagfsveeal filqsishft CAC81917hrdgarlhig ts.ptppqfe qaeaqlrcic dagfsveeal filqsishft P51561yrdggkihag tr.psesqfe tseqqlqflc dagfslsciavyalssiahft ZP_00132379yrdggkihag tr.psesqfe tseqqlqflc dagfslsciavyalssiahft AAD12754yrdggkihag tr.psancifetseqqlqflc dagftltqav yalssiahft P04483hrdgakvhlg tr.ptekqye tlencilaflcqqgfslenal yalsavghft A26948hrdgakvhlg tr.ptekqye tlenqlafya nkvfh~~~~~ ~~~~~~~~~~ CAC80726hrdaakihlg tr.pspecifetveacilaflceqgfsleeal ytlgvvshft P0ACT4YRDGAKVHLG TR.PDEKQYD TVETQLRFMT ENGFSLRDGL YAISAVSHFT ZP_01567051hrdgarlhag st.pdldave rirpkiaylv raglteqeag mamlaagqft NP_824556yrdgakvfsg srftgtehav qleaslrslv eagfdlpqav ratstayfft151                                                200 ZP_01558383vgwvleeqae aeretd.... .ttlpdtaeh p..llaqgwa alrerggdea YP_772551vgwvleeqae aeretd.... .ttlpdtaeh p..llaqgwt alrerggdea YP_620166vgwvleeqar pdgdtd.... .allpdaaey p..lfaqgwa alrersgdea EAY62734vgwvleeqar pdgdad.... .allpdaaey p..lfakgwa alrersgdea YP_368094vgwvleeqae ssdeaa.... .aplpdaaey p..llakgwa alrgrsgdda AAP93923vgwvleeqqm qpdda..... .lneadrrry p..llcggwe klqdkgadal AAW66496vgwvleeqqe eirsdp.... .pfeadptiy p..lmlqgvn tlqnmnaddi CAA24908vgavleeqag dsesgergg. ..tveqapls p..llraaid afdeagpdaa P03038vgavleeqag dsdagergg. ..tveqapls p..llraaid afdeagpdaa ABS19067vgavleeqag dsdagergg. ..tieqa... p..llravid tfdeagpdav NP_387462vgavleqqas eadaeerged qlttsastmp a..rlqsamk ivyeggpdaa NP_387455vgavleqqas eadaeerged qlttsastmp a..rlqsamk ivyeggpdaa AAR96033vgavleqqas eadaeerged qlttsastmp a..rlqsamk ivyeggpdaa NP_511232vgavleqqas eadaeerged qlttsastmp a..rlqsamk ivyeggpdaa AAW83818vgavleqqas eadaeerged qlttsastmp a..rlqsamk ivyeggpdaa AAD25094vgsvleqqas ..dadervpd rpdvseqaps s..flhdlfh eletdgmdaa AB014708vgsvleqqas ..dadervpd rpdvseqaps s..flhdlfh eletdgmdaa P51560vgsvleqqas ..dadervpd rpdvseqaps s..flhvlfh eletdgmdaa AAD25537vgsvleqqas ..nandrmsd ksdvseqaps s..flhdlfh eletdgmdap YP_001220607tgsyseqqaa ledsserkqa skeapaq.ps q..flshafd tfdaegadfa YP_001370475vgsaleqqa. ..dihesvpg ..daysitat s..eiaqaia ildadgaenl P21337lgavleeq.. atnqien..n hvid...aap p..llqeafn iqartsaema AAA98409lgavleeq.. atnqien..n hvid...aap p..llqeafn iqartsaema CAC81917lgavleeq.. atnptey..n tvmd...avp p..llqeafn vqtrttaeta P51561lgsvletqeh qesqker..e kvetdtvayp p..lltqava imdsdngdaa ZP_00132379lgsvletqeh qesqker..e kvetdtvayp p..lltqava imdsdngdaa AAD12754lgsvletqeh qesqker..e kvpkteinyp p..lltqaid imdsdngeaa P04483lgcvledgeh qvakeer..e tpttdsm..p p..llrqaie lfdhqgaepa A26948 ~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ CAC80726lgsvleerey leamrdd..d paihaam..p p..lltkale imeqdtgekp POACT4LGAVLEQQEH TAALTDR..P AAPDENL..P P..LLREALQ IMDSDDGEQA ZP_01567051igcvleqqaa qgrgaeepar rdaaddrp.. .....rtsga alapidpgva NP_824556lgfvteeqgv eplpgerreg ydvderaarm adfplaaaag aeifqnyeeg201                                  237 ZP_01558383fergvalivd gararla.ar rrgg~~~~~~ ~~~~~~~ YP_772551fergvalivd gararla.ar qrgg~~~~~~ ~~~~~~~ YP_620166fergiawivd gararla.ar rag~~~~~~~ ~~~~~~~ EAY62734fergiawivd gararla.ar rag~~~~~~~ ~~~~~~~ YP_368094fergvawivd gararla.ar erg~~~~~~~ ~~~~~~~ AAP93923feaglrllvd gaeaaltnan nhgaqs~~~~ ~~~~~~~ AAW66496fengirmvii gaerqldikm qt~~~~~~~~ ~~~~~~~ CAA24908feqglavivd glakrrlvvr nvegprkgdd ~~~~~~~ P03038feqglavivd glakrrlvvr nvegprkgdd ~~~~~~~ ABS19067felglavivd glakrrlvar niqgprkgdd ~~~~~~~ NP_387462ferglaliig gleqvrlspa sspagrtnlv lalaags NP_387455ferglaliig gleqvrlspa sspagrtnlv lalaags AAR96033ferglaliig glersacais ll~~~~~~~~ ~~~~~~~ NP_511232ferglaliig glekmrlttn dievlknvde ~~~~~~~ AAW83818ferglaliig glersacais ll~~~~~~~~ ~~~~~~~ AAD25094fnfgldslia gferlrss.. ttd~~~~~~~ ~~~~~~~ AB014708fnfgldslia gferlrss.. ttd~~~~~~~ ~~~~~~~ P51560fnfgldslia gferlraavl atd~~~~~~~ ~~~~~~~ AAD25537fnfgldslia gfeqlrls.. ttd~~~~~~~ ~~~~~~~ YP_001220607feygldalis glemkkatk~ ~~~~~~~~~~ ~~~~~~~ YP_001370475fdfglmllvd glerhrqs~~ ~~~~~~~~~~ ~~~~~~~ P21337fhfglkslif gfsaqldekk htpiedgnk~ ~~~~~~~ AAA98409fhfglkslif gfsaqldekk htpiedgnk~ ~~~~~~~ CAC81917fhfglrsliv gfsaqlde.k ymsiqgnnk~ ~~~~~~~ P51561flfvldvmis gletvlksak ~~~~~~~~~~ ~~~~~~~ ZP_00132379flfvldvmis gletvlksak ~~~~~~~~~~ ~~~~~~~ AAD12754flfvldvmis gletvinnhh ~~~~~~~~~~ ~~~~~~~ P04483flfgleliic glekqlkces gs~~~~~~~~ ~~~~~~~ A26948 ~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~ ~~~~~~~ CAC80726 flfgleviil gleakqkqkk gnqe~~~~~~ ~~~~~~~P0ACT4 FLHGLESLIR GFEVQLTALL QIVGGDKLII PFC~~~~ ZP_01567051fefglglivd glrrrvdra~ ~~~~~~~~~~ ~~~~~~~ NP_824556 feeglrlvia giearygir~~~~~~~~~~~ ~~~~~~~

Amino acid positions having relatively conserved amino acidsubstitutions between family members were considered for harvestingdiversity. In addition, positions were chosen for variation based onspacing to limit the number of changes in a pair of overlappingoligonucleotides. A summary of the library is shown in Table 11. Theobjective of this library was to recover hits improved for reactivity toeither ethametsulfuron or chlorsulfuron.

TABLE 11 Residue Position 52 55 62 69 73 76 79 83 85 88 93 96 98 101 102109 110 114 117 120 125 L1-9 Backbone L L R P E Q L A S C H G K L G Q YE L L F Sequence Shuffling L L D L A A F A A C H A K I G H F D I L FDiversity M M E P D D L N G R Y G R L R N V E L M L K E Q V S N S S Q YR V Y R R S Residue Position 129 130 137 140 145 149 162 167 170 175 181183 189 190 193 197 L1-9 Backbone N A V F V Q T P L E G E G L I LSequence Shuffling D A A F A Q Q P F D G D G I I A Diversity E G I Y V RT S I E S E V L L F H L L N G V M I N V M V L Q

Assembly of the L4 library synthetic oligonucleotides was done as forthe previous libraries, except that two sets of oligonucleotide poolswere used. First, multiple oligonucleotides representing diversity at asingle oligonucleotide annealing location are pooled (“Group” in Table12). Next, an equal volume of each group of oligos is pooled torepresent the novel L4 diversity. Likewise, oligonucleotidesrepresenting the L1-9 backbone sequence were pooled (Table 13). The L4assembly reaction was carried out by spiking the oligonucleotidediversity pool into the L1-9 backbone pool at an approximately 1:3ratio.

TABLE 12 SEQ Oligo Sequence Group ID L4:01TATTGGCATGTAAAAAATAAGCGAGCTCTGWTGGA 1 987 CGCCWTG L4:02TATTGGCATGTAAAGAATAAGVGCGCTCTGWTGGA 1 988 CGCCWTG L4:03GCCATTGAGATGCTCGATARACACGCCACTCACTT 2 989 TTGCCYC L4:04GCCATTGAGATGCTCGATGAKCACGCCACTCACTT 2 990 TTGCCYC L4:05TTAGAAGGGGMWAGCTGGCAAGATTTTBTTCGTAA 3 991 TAACGCA L4:06TTAGAAGGGGMWAGCTGGCAAGATTTTBTTCGTAA 3 992 TAACART L4:07TTAGAAGGGGMWAGCTGGAGGGATTTTBTTCGTAA 3 993 TAACGCA L4:08TTAGAAGGGGMWAGCTGGAGGGATTTTBTTCGTAA 3 994 TAACART L4:09TTAGAAGGGGMWAGCTGGGMTGATTTTBTTCGTAA 3 995 TAACGCA L4:10TTAGAAGGGGMWAGCTGGGMTGATTTTBTTCGTAA 3 996 TAACART L4:11AAAARTATGAGAHGTGCTTTACTAAGTYACCGCGA 4 997 TGSAGCA L4:12AAAGSAATGAGAHGTGCTTTACTAAGTYACCGCGA 4 998 TGSAGCA L4:13ARAGTATGCTCCRGGACAGGATTTACAGAAAAACA 5 999 AKTTGAA L4:14ARAGTATGCTCCRGGACAGGATTTACAGAAAAACA 5 1000 ATACGAA L4:15ARAGTATGCTCCRGGACAGGATTTACAGAAAAAMA 5 1001 TKTTGAA L4:16ARAGTATGCTCCRGGACAGGATTTACAGAAAAAMA 5 1002 TTACGAA L4:17ARAGTATGCMTCRGGACAGGATTTACAGAAAAACA 5 1003 AKTTGAA L4:18ARAGTATGCMTCRGGACAGGATTTACAGAAAAACA 5 1004 ATACGAA L4:19ARAGTATGCMTCRGGACAGGATTTACAGAAAAAMA 5 1005 TKTTGAA L4:20ARAGTATGCMTCRGGACAGGATTTACAGAAAAAMA 5 1006 TTACGAA L4:21ACTGCTGAMAATTCAVTTGCCTTTMTGTGCCAACA 6 1007 AGGTTTK L4:22ACTGCTGAMAATTCAVTTGCCTTTTACTGCCAACA 6 1008 AGGTTTK L4:23ACTGCTAGGAATTCAVTTGCCTTTMTGTGCCAACA 6 1009 AGGTTTK L4:24ACTGCTAGGAATTCAVTTGCCTTTTACTGCCAACA 6 1010 AGGTTTK L4:25TCACTAGAGVACGSATTATATGCAATGCAAGCTGC 7 1011 ATGTATT L4:26TCACTAGAGVACGSATTATATGCAATGCAAGCTVT 7 1012 CTGTATT L4:27TCACTAGAGSAAGSATTATATGCAATGCAAGCTGC 7 1013 ATGTATT L4:28TCACTAGAGSAAGSATTATATGCAATGCAAGCTVT 7 1014 CTGTATT L4:29TWCACTTTAGGTTGCGYATTGCTCGATCAAGAGTT 8 1015 GCAAGTC L4:30TWCACTTTAGGTTGCGYATTGCTCGATCGTGAGTT 8 1016 GCAAGTC L4:31GCTAAAGAAGAAAGGGAAACACCTCAAACTGATAG 9 1017 TATGYCT L4:32GCTAAAGAAGAAAGGGAAACACCTACTACTGATAG 9 1018 TATGYCT L4:33CCATTAWTKCGACAAGCTTTGAATTTAAAGGATCA 10 1019 CCAARGC L4:34CCATTAWTKCGACAAGCTTTGGAWTTAAAGGATCA 10 1020 CCAARGC L4:35GCAGRWCCAGCCTTCTTATTCGKGVTTGAATTGVT 11 1021 KATATGC L4:36GGAHTTGAAAAACAACTTAAATGTGAAAGTGGGTC 12 1022 TTAA L4:37GGAGCTGAAAAACAACTTAAATGTGAAAGTGGGTC 12 1023 TTAA L4:38TYTATCGAGCATCTCAATGGCCAWGGCGTCCAWCA 13 1024 GAGCTCG L4:39MTCATCGAGCATCTCAATGGCCAWGGCGTCCAWCA 13 1025 GAGCTCG L4:40TYTATCGAGCATCTCAATGGCCAWGGCGTCCAWCA 13 1026 GAGCGCB L4:41MTCATCGAGCATCTCAATGGCCAWGGCGTCCAWCA 13 1027 GAGCGCB L4:42TTGCCAGCTWKCCCCTTCTAAGRGGCAAAAGTGAG 14 1028 TGGCGTG L4:43CCTCCAGCTWKCCCCTTCTAAGRGGCAAAAGTGAG 14 1029 TGGCGTG L4:44AKCCCAGCTWKCCCCTTCTAAGRGGCAAAAGTGAG 14 1030 TGGCGTG L4:45TAAAGCACDTCTCATAYTTTTTGCGTTATTACGAA 15 1031 VAAAATC L4:46TAAAGCACDTCTCATTSCTTTTGCGTTATTACGAA 15 1032 VAAAATC L4:47TAAAGCACDTCTCATAYTTTTAYTGTTATTACGAA 15 1033 VAAAATC L4:48TAAAGCACDTCTCATTSCTTTAYTGTTATTACGAA 15 1034 VAAAATC L4:49TCCTGTCCYGGAGCATACTYTTGCTSCATCGCGGT 16 1035 RACTTAG L4:50TCCTGTCCYGAKGCATACTYTTGCTSCATCGCGGT 16 1036 RACTTAG L4:51GGCAABTGAATTKTCAGCAGTTTCAAMTTGTTTTT 17 1037 CTGTAAA L4:52GGCAABTGAATTCCTAGCAGTTTCAAMTTGTTTTT 17 1038 CTGTAAA L4:53GGCAABTGAATTKTCAGCAGTTTCGTATTGTTTTT 17 1039 CTGTAAA L4:54GGCAABTGAATTCCTAGCAGTTTCGTATTGTTTTT 17 1040 CTGTAAA L4:55GGCAABTGAATTKTCAGCAGTTTCAAMATKTTTTT 17 1041 CTGTAAA L4:56GGCAABTGAATTCCTAGCAGTTTCAAMATKTTTTT 17 1042 CTGTAAA L4:57GGCAABTGAATTKTCAGCAGTTTCGTAATKTTTTT 17 1043 CTGTAAA L4:58GGCAABTGAATTCCTAGCAGTTTCGTAATKTTTTT 17 1044 CTGTAAA L4:59ATATAATSCGTBCTCTAGTGAMAAACCTTGTTGGC 18 1045 ACAKAAA L4:60ATATAATSCTTSCTCTAGTGAMAAACCTTGTTGGC 18 1046 ACAKAAA L4:61ATATAATSCGTBCTCTAGTGAMAAACCTTGTTGGC 18 1047 AGTAAAA L4:62ATATAATSCTTSCTCTAGTGAMAAACCTTGTTGGC 18 1048 AGTAAAA L4:63CAATRCGCAACCTAAAGTGWAAATACATGCAGCTT 19 1049 GCATTGC L4:64CAATRCGCAACCTAAAGTGWAAATACAGABAGCTT 19 1050 GCATTGC L4:65TGTTTCCCTTTCTTCTTTAGCGACTTGCAACTCTT 20 1051 GATCGAG L4:66TGTTTCCCTTTCTTCTTTAGCGACTTGCAACTCAC 20 1052 GATCGAG L4:67CAAAGCTTGTCGMAWTAATGGAGRCATACTATCAG 21 1053 TTTGAGG L4:68CAAAGCTTGTCGMAWTAATGGAGRCATACTATCAG 21 1054 TAGTAGG L4:69GAATAAGAAGGCTGGWYCTGCGCYTTGGTGATCCT 22 1055 TTAAATT L4:70GAATAAGAAGGCTGGWYCTGCGCYTTGGTGATCCT 22 1056 TTAAWTC L4:71TTTAAGTTGTTTTTCAADTCCGCATATMABCAATT 23 1057 CAABCMC L4:72TTTAAGTTGTTTTTCAGCTCCGCATATMABCAATT 23 1058 CAABCMC L1:50GGGAACTTCGGCGCGCCTTAAGACCCACTTTCACA 24 1059

TABLE 13 SEQ Oligo Sequence ID L1-9:01TATTGGCATGTAAAGAATAAGCGTGCTCTGTTGGAC 1060 GCCCTG L1-9:02GCCATTGAGATGCTCGATCGTCACGCCACTCACTTT 1061 TGCCCT L1-9:03TTAGAAGGGGAAAGCTGGCAAGATTTTCTCCGTAAT 1062 AATGCA L1-9:04AAATCAATGAGATGCGCTTTACTAAGTCATCGCGAT 1063 GGGGCA L1-9:05AAGGTATGTCTTGGTACAGGATTCACAGAAAAACAG 1064 TACGAA L1-9:06ACTGCTGAAAATAGTTTGGCCTTTCTGTGCCAACAA 1065 GGTTTC L1-9:07TCACTAGAGAATGCTTTATATGCAATGCAAGCTGTC 1066 TGTATC L1-9:08TTCACTTTAGGTTGCGTTTTGCTGGATCAAGAGCTC 1067 CAAGTC L1-9:09GCTAAAGAAGAAAGGGAAACACCTACTACTGATAGT 1068 ATGCCC L1-9:10CCATTATTGCGACAAGCTTTGGAATTAAAAGATCAC 1069 CAAGGG L1-9:11GCAGAGCCAGCCTTCTTATTCGGATTGGAATTGATA 1070 ATATGC L1-9:12GGATTGGAAAAACAACTTAAATGTGAAAGTGGGTCT 1071 TAA L1-9:13ACGATCGAGCATCTCAATGGCCAGGGCGTCCAACAG 1072 AGCACG L1-9:14TTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGT 1073 GGCGTG L1-9:15TAAAGCGCATCTCATTGATTTTGCATTATTACGGAG 1074 AAAATC L1-9:16TCCTGTACCAAGACATACCTTTGCCCCATCGCGATG 1075 ACTTAG L1-9:17GGCCAAACTATTTTCAGCAGTTTCGTACTGTTTTTC 1076 TGTGAA L1-9:18ATATAAAGCATTCTCTAGTGAGAAACCTTGTTGGCA 1077 CAGAAA L1-9:19CAAAACGCAACCTAAAGTGAAGATACAGACAGCTTG 1078 CATTGC L1-9:20TGTTTCCCTTTCTTCTTTAGCGACTTGGAGCTCTTG 1079 ATCCAG L1-9:21CAAAGCTTGTCGCAATAATGGGGGCATACTATCAGT 1080 AGTAGG L1-9:22GAATAAGAAGGCTGGCTCTGCCCCTTGGTGATCTTT 1081 TAATTC L1-9:23TTTAAGTTGTTTTTCCAATCCGCATATTATCAATTC 1082 CAATCC L1-9:24GGGAACTTCGGCGCGCCTTAAGACCCACTTTCACA 1083

The assembly reaction products were cloned into the pVER7314 backboneand transformed into tester strain E. coli KM3. To carry out librarydiversity analysis, DNA preps from 96 colonies grown on LB+Cb only(representing no repressor positive selection bias) were subjected tosequence analysis. These data showed approximately 30% of the clonesrecovered were unaltered L1-9 backbone and the remaining clones hadapproximately one to two targeted changes per clone. Additionalnon-targeted residue changes were recovered in the mutated population,either due to PCR errors or from poor quality oligonucleotidesincorporated into the assembly reactions.

I. Library L4 Screening

Approximately 20,000 clones arising from the repressor prescreen weretested for activation by 0, 0.2 and 1 μg/ml concentrations ofethametsulfuron using the M9 assay plates. Surprisingly, over 100 hitswere observed from the 0.2 μg/ml ethametsulfuron treatment. Theseputative hits were re-arrayed in 96-well format and re-tested forβ-galactosidase induction by 0, 0.2 and 1 ppm ethametsulfuron using aliquid culture based assay system. FIG. 4 shows relative β-galactosidaseactivities of 45 exemplary putative library L4 hit clones 97-142 against0, 0.2 and 1 ppm ethametsulfuron. Cultures grown in 96-well format weresubcultured into fresh LB with inducer at the indicated concentrationand grown overnight and then processed for the enzyme assay. Fordetection of induced activity, 5 μl of perforated cell mixture was used.For detection of background activity, 25 μl of cells were used such thatdetectable activity could be measured in the same time frame for alltreatments. Background activity values were multiplied by ten to bringthem into the range of the graph. The numbers below each sample refersto the library clone number. The latter part of the graph contains thecontrols 1st round hit L1-9 as well as wt TetR.

DNA sequences for all 142 putative hit clones were determined and thetranslated polypeptides aligned. After assigning each polypeptide in thealignment with a relative ethametsulfuron response, patterns of aminoacid incorporation at varied or mutated residues associated with high orlow response activity and high or low uninduced activity wereidentified. The most significant findings from this analysis were: C138Gor L170V mutations were heavily favored in the top clones L4-59, -89,-110, -116, -118, -120, -124, -129, -133, -139, -140 and -142; and K108Qwas heavily incorporated in clones with the highest activity at thelowest dose of 0.2 ppm, but these clones showed somewhat leakybackground (e.g., L4-98, -106, -113, -126, -130, and -141). Results fromclone L4-18 having the K108Q shows another possible interesting mutationof L55M. This clone is induced to a high level with 0.2 ppm Es, but doesnot show the associated high background activity typically observed forK108Q-containing clones. The L55M mutation may have increased repressoractivity. It is of interest that none of these changes other than L55Mwere designed diversity—all were derived from false incorporation ofnucleotides during library assembly and few of these changes wererepresented in the unselected clone population.

J. Third Round Library Designs and Screening

Library L6: Shuffling for Enhanced Chlorsulfuron Response

Since clones L2-14 and L2-18 had the best chlorsulfuron activity profilefrom library L2, their amino acid diversity was used as the basis forthe next round of shuffling. In addition to the diversity provided bythese backbone sequences, additional residue changes thought to enhancepacking of chlorsulfuron based on the 3D model predictions wereincluded. New amino acid positions targeted were 67, 109, 112 and 173(see, Table 14). Substitution of Gln (Q) at position 108 and Val (V) atposition 170 were shown to likely be important changes in library L4 forgaining enhanced SU responsiveness and so were varied here as well. Asummary of the diversity chose is shown in Table 14. Theoligonucleotides designed and used to generate library 6 are shown inTable 15.

Library L6 was assembled, rescued, ligated into pVER7314, transformedinto E. coli KM3 and plated out onto LB carbenicillin/kanamycin, andcarbenicillin only control media as before. Library plates were thenpicked into 42 384-well microtiter plates (˜16,000 clones) containing 60μl LB carbenicillin (Cb) broth per well. After overnight growth at 37°C. the cultures were stamped onto M9 assay plates containing no inducer,0.2 ppm, and 2.0 ppm chlorsulfuron as test inducer. Following incubationat 30° C. for ˜48 hrs, putative hits responding to chlorsulfurontreatment as determined by increased blue colony color were re-arrayedinto six 96-well microtiter plates and used to stamp a fresh set of M9assay plates to confirm the above results. For a more detailed analysisof the relative induction by chlorsulfuron, digital photographs weretaken of the plates after various time points of incubation at 30° C.and colony color intensity measured using the digital image analysisfreeware program ImageJ (Rasband, US National Institutes of Health,Bethesda, Md., USA, rsb.info.nih.gov/ij/, 1997-2007). Using theseresults enabled ranking of clones in multiplex format by backgroundactivity (no inducer), activation with low or high level inducerapplication (blue color with inducer), and fold activation (activationdivided by background). Activation studies using 0.2 μg/ml chlorsulfuronas inducer for the top set of clones shows an approximately 3 foldimprovement in activation while obtaining lower un-induced levels ofexpression (Table 12). In addition to this analysis, DNA sequenceinformation for most clones (490 clones) was obtained and the deducedpolypeptides aligned with each other as well as with their correspondingactivity information. From this analysis sequence-activity relationshipswere derived (Table 12). Residues biased for improved activity areindicated in larger bold type. Briefly, C at position 100, and Q atpositions 108 and 109 strongly correlated with activation, while R atposition 138, L at position 170, and A or G at position 173 were highlypreferred in clones with the lowest background activity. Though somepositions were strongly biased, i.e., observed more frequently in theselected population, the entirety of introduced diversity was observedin the full hit population. This information will aid in the design offurther libraries to improve responsiveness to chlorsulfuron.

TABLE 14 Sequence Amino acid residue position Name 60 64 67 82 86 100105 108 109 112 113 116 134 138 139 Library A M N C Q M S M M G NDiversity Q Y T W K L T Q V R V F Q A L H G I V wt reference L H F N F HP K Q T L Q L G H L2-14 M A F N M C W K Q T A M V R V L2-18 M Q F T M WW K Q T A Q M R N L6-1B03 M A I N M C W Q Q A A M V R V L6-2C09 M Q Y TM C W Q L T A Q M R V L6-2D07 M Q F T M C W Q Q T A M M R V L6-3H02 M AY T M C W Q H S A M V R V L6-4D10 M Q Y N M C W K Q S A M V R V L6-5F05M A I N M C W Q Q A A Q V R V L6-5G06 M Q Y N M C W Q Q T A Q V R VL6-5H06 M Q I N M C W K Q T A M V R V L6-5H12 M A Y N M C W K Q T A Q MR V L6-6F07 M A L T M C W Q Q S A M M R V Bias in top none Y N C Q Qnone none V R V population Amino acid Sequence residue position Name 147151 164 170 173 174 177 178 0.2 ppm Library S L G L 0.2 48 hr/ DiversityL A A W ppm Control Control V V 48 hr 84 hr 84 hr wt reference E H D L AI F D 5.2 5.3 1.0 L2-14 F S A L A L K D 11.8 6.6 1.8 L2-18 F L A L A W KD 5.9 5.7 1.0 L6-1B03 F S A L A W K D 30.0 6.6 4.6 L6-2C09 F L A L A W KD 13.6 5.2 2.6 L6-2D07 F S A V A W K D 20.0 5.8 3.4 L6-3H02 F S A V A WK V 15.8 5.6 2.8 L6-4D10 F S A L A W K D 18.4 5.0 3.7 L6-5F05 F L A L AW K D 22.0 5.4 4.1 L6-5G06 F L A L G W K D 34.4 7.0 4.9 L6-5H06 F L A VA W K D 13.7 5.1 2.7 L6-5H12 F L A V A W K D 23.7 5.7 4.2 L6-6F07 F S AL A W K D 11.6 5.1 2.3 Bias in top none L A/G W D population

TABLE 15 Oligo Sequence SEQ ID L6:1TATTGGCATGTAAAAAATAAGCGAGCTCTGCTCGACGC 1084 CTTA L6:2GCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCAT 1085 ATGC L6:3ATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATA 1086 AAAA L6:4TTTAAGTTGTTTTTCTAATCCGCATATGATCAATTCAA 1087 GGCC L6:5TTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAM 1088 TGCT L6:6TAAAGCACATCTCATACTTTTAGCAKTATTACGTAAAA 1089 AATC L6:7TTGCCAGCTTTCCCCTTCTAAAGGGCAMAHGTGAGTTG 1090 CGTG L6:8TTGCCAGCTTTCCCCTTCTAAAGGGCAATAGTGAGTTG 1091 CGTG L6:9GAATAAGAAGGCTGGCTCTGCACCTTGGTGATCCTTTA 1092 ATTC L6:10GCCATTGAGATGATGGATAGGCACGCAACTCACTATTG 1093 CCCT L6:11RSTGCTGAAAATATGTTAGCCTTTTTATGCCAACAAGG 1094 TTTT L6:12TTTACTTTAGGTTGCGTATTGTTTGATCAAGAGCTCCA 1095 AGTC L6:13TGTTTCCCTTTCTTCTTTAGCGACTTGGAGCTCTTGAT 1096 CAAA L6:14GCCATTGAGATGATGGATAGGCACGCAACTCACDTKTG 1097 CCCT L6:15GCCATTGAGATGATGGATAGGCACCAAACTCACDTKTG 1098 CCCT L6:16GCCATTGAGATGATGGATAGGCACCAAACTCACTATTG 1099 CCCT L6:17AAAAGTATGAGATGTGCTTTACTAAGCCATCGCGATGG 1100 AGCA L6:18AAAGTATGKTTAGGTACACGCTGGACAGAAMAACAWTA 1101 TGAA L6:19AAAGTATGKTTAGGTACACGCTGGACAGAAMAAWTGTA 1102 TGAA L6:20RSTGCTGAAAATCAATTAGCCTTTTTATGCCAACAAGG 1103 TTTT L6:21TCACTAGAGAATGCATTATATGCARTGAGTGCGTGGRG 1104 GGTG L6:22TCACTAGAGAATGCATTATATGCARTGAGTGCGTGGRG 1105 GAAC L6:23TTTACTTTAGGTTGCGTATTGTTTGATCAAGAGAGCCA 1106 AGTC L6:24GCTAAAGAAGAAAGGGAAACACCTACTACTGCTAGTAT 1107 GCCG L6:25CCATTAKTGCGACAAGBTTKGGAATTAAAGGATCACCA 1108 AGGT L6:26CCATTAGCCCGACAAGBTTKGGAATTAAAGGATCACCA 1109 AGGT L6:27GGATTAGAAAAACAACTTAAATGCGAAAGTGGGTCTT 1110 AA L6:28CCTATCCATCATCTCAATGGCTAAGGCGTCGAGCAGAG 1111 CTCG L6:29TTGCCAGCTTTCCCCTTCTAAAGGGCAMAHGTGAGTTT 1112 GGTG L6:30TTGCCAGCTTTCCCCTTCTAAAGGGCAATAGTGAGTTT 1113 GGTG L6:31GCGTGTACCTAAMCATACTTTTGCTCCATCGCGATGGC 1114 TTAG L6:32GGCTAACATATTTTCAGCASYTTCATAWTGTTKTTCTG 1115 TCCA L6:33GGCTAATTGATTTTCAGCASYTTCATAWTGTTKTTCTG 1116 TCCA L6:34GGCTAACATATTTTCAGCASYTTCATACAWTTKTTCTG 1117 TCCA L6:35GGCTAATTGATTTTCAGCASYTTCATACAWTTKTTCTG 1118 TCCA L6:36CAATACGCAACCTAAAGTAAACACCCYCACAGCACTCA 1119 YTGC L6:37CAATACGCAACCTAAAGTAAAGTTCCYCACAGCACTCA 1120 YTGC L6:38TGTTTCCCTTTCTTCTTTAGCGACTTGGCTCTCTTGAT 1121 CAAA L6:39CMAAVCTTGTCGCAMTAATGGCGGCATACTAGCAGTAG 1122 TAGG L6:40CMAAVCTTGTCGGGCTAATGGCGGCATACTAGCAGTAG 1123 TAGG L6:41GGGAACTTCGGCGCGCCTTAAGACCCACTTTCGCA 1124K. Library L7: Shuffling for Enhanced Ethametsulfuron Response

The choice of parents to represent the amino acid residue diversity forlibrary L7 was based on the conclusions of library L4 analysis—namelyincorporation of mutations K108Q, C138G and L170V. Clones were alsochosen to bring in other changes that occurred at a much lower frequencyin L4, but may have been contributing to activity. These residues areL55M, N129H, V137A and F140Y. In addition to family diversity, otherresidue modifications were introduced at amino acid positions 67, 109,112, 117, 131 and 173 based on structural modeling. This information issummarized in Table 14 showing L7 diversity summary. Also shown in Table16 is a sequence alignment the top 10 performing L7 hits limited to thedifferences between the hits and wt TetR. Activity was determined usingimage analysis of colony color (ImageJ software) on M9 assay platescontaining 0, 0.02 or 0.2 ppm ethametsulfuron. At the bottom of Table 16is a summary of the sequence-activity relationship analysis for theentire data set derived from more than 300 clones, with the stronglybiased positions shown in larger bolded type. Even though some positionswere strongly biased, i.e., observed more frequently in the selectedpopulation, e.g., M at position 55, the entirety of introduced diversitywas observed in the full hit population.

TABLE 16 Amino acid residue position Sequence 55 64 67 85 86 100 104 105108 109 112 113 116 117 129 131 134 135 137 138 139 140 147 151 170 173174 177 wt refer- L H F S F H R P K Q T L Q L N L L S V G H F E H L A IF ence L7 diversity M M Q M S M N M A G F L G L Y K L T L H L V C Y A AF Q A V V L H G V L7-1C03-A05 M A V I M C G F Q Q — A S — H — M Q A G I— L L V — L N L7-1C07-A06 M A Y — M C G F Q Q — A S — H — M Q A G I — LL V — L K L7-1F08-A11 M A Y — M C G F Q Q — A S — — — M Q — G I Y L L V— L K L7-1G06-B02 M A Y — M C G F Q Q — A S M — — M Q — G I Y L L V — LK L7-2C11-B11 M A Y — M C G F Q Q — A S M — M M Q A G I — L L V V L KL7-2D08-C02 M A Y — M C G F Q Q — A S — H — M Q — G I Y L L V — L KL7-3A10-C09 M A Y — M C G F Q Q — A S — H — M Q — G I Y L L V — L KL7-3C08-C10 M A Y — M C G F Q Q G A S M — M M Q A G I Y L L V — L KL7-3E03-D01 M A Y — M C G F Q Q — A S — H M M Q A G I Y L L V — L KL7-3E04-D02 M A Y — M C G F Q Q S A S — H M M Q A G I — L L V — L KBias in top M Y Q Q N G V population

The L7 library was constructed as for Library L1 using the set ofoligonucleotides shown below in Table 17.

TABLE 17 Oligo SEQ ID Oligo sequence ID L7:01TATTGGCATGTAAAAAATAAGCGAGCTCTGCTCGACGCA 1125 WTG L7:02GCCATTGAGATGCTGGATAGGCACGCGACTCACDTSTGC 1126 CCT L7:03GCCATTGAGATGCTGGATAGGCACGCGACTCACTATTGC 1127 CCT L7:04TTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAAC 1128 GCT L7:05AAAAGTATGAGATGTGCTTTACTAAGTCATCGCGATGGA 1129 GCA L7:06AAAGTATGTTTAGGTACAGGCTTTACAGAAMAGMTGTAT 1130 GAA L7:07AAAGTATGTTTAGGTACAGGCTTTACAGAAMAGCAMTAT 1131 GAA L7:08RSTGCCGAAAATAGTMTGGCCTTTTTATGCCAACAAGGT 1132 TTT L7:09TCACTAGAGMACGCAMTGTATGCAATGCAGGCTGYTKGT 1133 ATT L7:10TWTACTTTAGGTTGCGTATTGTTGGATCAAGAGCTTCAA 1134 GTC L7:11GCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATG 1135 CCG L7:12CCATTAGCTCGACAAGBTCTGGAATTAAAGGATCACCAA 1136 GGT L7:13CCATTASTCCGACAAGBTCTGGAATTAAAGGATCACCAA 1137 GGT L7:14GCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATA 1138 TGC L7:15GGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCTTAA 1139 L7:16CCTATCCAGCATCTCAATGGCCAWTGCGTCGAGCAGAGC 1140 TCG L7:17TTGCCAGCTTTCCCCTTCTAAAGGGCASAHGTGAGTCGC 1141 GTG L7:18TTGCCAGCTTTCCCCTTCTAAAGGGCAATAGTGAGTCGC 1142 GTG L7:19TAAAGCACATCTCATACTTTTAGCGTTATTACGTAAAAA 1143 ATC L7:20GCCTGTACCTAAACATACTTTTGCTCCATCGCGATGACT 1144 TAG L7:21GGCCAKACTATTTTCGGCASYTTCATACAKCTKTTCTGT 1145 AAA L7:22GGCCAKACTATTTTCGGCASYTTCATAKTGCTKTTCTGT 1146 AAA L7:23ATACAKTGCGTKCTCTAGTGAAAAACCTTGTTGGCATAA 1147 AAA L7:24CAATACGCAACCTAAAGTAWAAATACMARCAGCCTGCAT 1148 TGC L7:25TGTTTCCCTTTCTTCTTTAGCGACTTGAAGCTCTTGATC 1149 CAA L7:26CAGAVCTTGTCGAGCTAATGGCGGCATACTATCAGTAGT 1150 AGG L7:27CAGAVCTTGTCGGASTAATGGCGGCATACTATCAGTAGT 1151 AGG L7:28GAATAAGAAGGCTGGCTCTGCACCTTGGTGATCCTTTAA 1152 TTC L7:29TTTAAGTTGTTTTTCTAATCCGCATATGATCAATTCAAG 1153 GCC L7:30GGGAACTTCGGCGCGCCTTAAGACCCACTTTCACA 1154

After transformation of the library into E. coli KM3 and plating onLB+Cb+Km the resulting colonies were reformatted into fifty-two 384-wellmicrotiter plates (˜20,000 colonies) and subsequently used to replicaplate onto M9 assay medium containing either 0 μg/ml, 0.02 μg/ml or 0.2μg/ml ethametsulfuron. After incubation at 30° C. for 48 hrs the plateswere observed and 326 hits responding to 0.02 μg/ml inducer wereidentified and re-arrayed into 96-well format. Following incubation at30° C. for 15, 24, 48 and 120 hours, digital images of the plates weretaken and the relative colony color information converted to numericaldata. DNA sequence analysis was carried out in parallel, and the twodata sets merged for calculation of sequence-activity relationships.Sequence data for the top ten clones as well as the summary of thesequence-activity relationships are shown in Table 16. The results ofsequence-activity relationship study revealed preferences impacting bothactivation and background activities of the putative ethametsulfuronrepressors (EsR's). For example, one significant finding from thislibrary was that modification L55M greatly reduced background activityand thus enhanced levels of fold activation. As seen from libraries L4and L6, K108Q and wt Q109 were preferred for activation. There was alsoa high degree of bias towards L170V related to activation. This isdifferent from the L170A or L170G bias seen in library L6, as thosemodifications had a strong correlation with lowering background activityin library L6. Finally, having a less dramatic effect on activation butnevertheless preferred is F67Y.

L. Induction Properties of Top L7 Hits in Liquid Cultures

Based on the performance of the re-arrayed hits, a second re-array wasdone and the clones tested for β-galactosidase activity in liquidculture along with wt TetR controls and selected 2^(nd) round hits tofurther analyze their performance and shuffling progress. Top hits fromthe L7 library were re-arrayed and tested in 96-well culture format forrelative induction by 0.02 and 0.2 ug/ml inducer or background activitywithout inducer (FIG. 5). Cultures were grown overnight and thensubcultured into fresh medium containing appropriate treatments.Following six hours of incubation the cells were processed for enzymeassay. For assay of induced activity 5 μl of perforated cell mixture wasused, for background activity 25 μl of cells were used such thatdetectable activity could be measured in the same time frame for alltreatments. Background activity values were multiplied by ten to bringthem into the range of the graph. The numbers below each sample refersto the final re-array well ID (vertical writing) and original re-arraywell ID (horizontal writing). The latter part of the graph contains thecontrols. Shown are 2nd round hits L4-89 and L4-120 as well as wt TetR.The final sample shows a control comprising wt TetR with 0.4 μM atc ascognate inducer for comparison. Results show that ten to fifteen of thetop hits have induced activity approaching that of wt TetR induced with0.4 μM atc. In addition, many of the hits have background activitiesnearly as low as wild type TetR. Some of the best hits have inductionratios with 0.2 ppm inducer (0.5 μM) approaching 70-80% of that of wtTetR (˜1200-fold). It is interesting that the hits performing best atthe low inducer concentration of 0.02 ppm (50 nM) also tended to havethe higher background activity indicating that they are less tightlybound to tet operator and more easily released with transient inducerbinding.

Comparison of induction activity between the 2^(nd) and 3^(rd) roundhits is striking, showing greater than 200-fold improvement. Consideringthis improvement, a single additional round of shuffling and screeningmay yield sulfonylurea repressors (SuRs) that are nearly as sensitive toethametsulfuron as the wt TetR is for tetracycline.

Summary

FIG. 9 provides a cumulative summary of the introduced diversity andobserved amino acids in active SuRs obtained from the screening assays.Even though some positions were strongly biased i.e., observed morefrequently in the selected population, as indicated by larger boldedtype, the entirety of introduced diversity was observed in the full hitpopulations.

M. Novel Diversity Through In Vitro Mutagenesis

Residues A64, M86, C100, G104, F105, Q108, A113, S116, M134, Q135, I139,Y140, L147, L151, V170, L174, and K177 of round 3 hit L7-A11 were eachmutagenized to all possible 20 amino acids to generate a set of 340clones. Each of the clones was replica plated onto M9 assay mediumcontaining 0, 5, 20 and 200 ppb ethametsulfuron. To assess relativeactivity of each of the mutants the plates containing ligand werephotographed following 18 hrs of incubation at 37° C. To determineleakiness of the repressor clones the plate having no ligand additionwas photographed after incubation for 24 hrs at 37° C. followed by 48hrs of incubation at 25° C. Quantitative measurements were made byscanning digital photographs of each colony for blue color using ImageJsoftware.

These data revealed that select substitutions at positions L60, A64,N82, M86, A113, S116, M134, L174, and K177 demonstrated an increase inethametsulfuron sensitivity relative to the parent clone L7-A11.

N. Fifth-Round Shuffling

Shuffling designed for improved ethametsulfuron sensitivity wasperformed. Library L13 (Table 18) was designed to incorporate noveldiversity generated by the in vitro mutagenesis experiment in Example 1Mthat had either positive or neutral effect on activity. In addition, thelibrary also incorporated diversity at selected cysteine residues in thebackbone as listed (Table 18). The predicted library size is 124,000members.

TABLE 18 Library L13 Residue 60 64 68 82 86 88 100 113 116 121 134 174177 195 203 L7-A11 L A C N M C C A S C M L K C C Diversity L A L K M N CA S T M L K C C F D C R R A M C F I H S A K N W G F R Y

The library was assembled from synthetic oligonucleotides listed inTable 19 using methodology as described previously in this example.

TABLE 19 SEQ Oligo Sequence ID L13:1TGGCACGTCAAGAACAAGCGAGCTCTGCTAGACGCTA 1159 TGGCC L13:2ATTGAGATGTTSGATAGGCACAAGACCCACTACTGTC 1160 CTTTG L13:3ATTGAGATGTTSGATAGGCACAAGACCCACTACCTGC 1161 CTTTG L13:4ATTGAGATGTTSGATAGGCACGMCACCCACTACTGTC 1162 CTTTG L13:5ATTGAGATGTTSGATAGGCACGMCACCCACTACCTGC 1163 CTTTG L13:6GAAGGGGAAAGCTGGCAAGACTTCTTGAGGAACAATG 1164 CTAAG L13:7GAAGGGGAAAGCTGGCAAGACTTCTTGAGGAACARGG 1165 CTAAG L13:8TCCAKGAGAAATGCTTTGCTCAGTCACCGTGATGGAG 1166 CCAAG L13:9GTCTGCCTAGGTACGGGCTTCACGGAGCAACAGTATG 1167 AAACT L13:10GTCGCTCTAGGTACGGGCTTCACGGAGCAACAGTATG 1168 AAACT L13:11GCTGAGAACTSKCTTGCCTTCCTGACACAACAAGGTT 1169 TCTCC L13:12ATGGAGAACTSKCTTGCCTTCCTGACACAACAAGGTT 1170 TCTCC L13:13CTTGAGAACGCCCTCTACGCATTTCAAGCTGTTGGGA 1171 TCTAC L13:14CTTGAGAACGCCCTCTACGCAGGTCAAGCTGTTGGGA 1172 CTCTA L13:15CTTGAGAACGCCCTCTACGCAATGCAAGCTGTTGGGA 1173 TCTAC L13:16ACTCTGGGTTGCGTCTTGCTGGATCAAGAGCTGCAAG 1174 TCGCT L13:17AAGGAGGAGAGGGAAACACCTACTACTGATAGTATGC 1175 CGCCA L13:18CTGGTTCGACAAGCTTACGAACTCCACGATCACCAAG 1176 GTGCA L13:19CTGGTTCGACAAGCTTACGAACTCARAGATCACCAAG 1177 GTGCA L13:20CTGGTTCGACAAGCTHTCGAACTCCACGATCACCAAG 1178 GTGCA L13:21CTGGTTCGACAAGCTHTCGAACTCARAGATGTGCACA 1179 CCAAG L13:22GAGCCAGCCTTCCTGTTCGGCCTTGAACTGATCATAW 1180 GTGGA L13:23TTGGAGAAGCAGCTGAAGTGTGAAAGTGGGTCTTAAT 1181 GATAG L13:24TTGGAGAAGCAGCTGAAGGCAGAAAGTGGGTCTTAAT 1182 GATAG L13:25GTGCCTATCSAACATCTCAATGGCCATAGCGTCTAGC 1183 AGAGC L13:26GTCTTGCCAGCTTTCCCCTTCCAAAGGACAGTAGTGG 1184 GTCTT L13:27GTCTTGCCAGCTTTCCCCTTCCAAAGGCAGGTAGTGG 1185 GTCTT L13:28GTCTTGCCAGCTTTCCCCTTCCAAAGGACAGTAGTGG 1186 GTGKC L13:29GTCTTGCCAGCTTTCCCCTTCCAAAGGCAGGTAGTGG 1187 GTGKC L13:30GAGCAAAGCATTTCTCMTGGACTTAGCATTGTTCCTC 1188 AAGAA L13:31GAGCAAAGCATTTCTCMTGGACTTAGCCYTGTTCCTC 1189 AAGAA L13:32GAAGCCCGTACCTAGGCAGACCTTGGCTCCATCACGG 1190 TGACT L13:33GAAGCCCGTACCTAGAGCGACCTTGGCTCCATCACGG 1191 TGACT L13:34GAAGGCAAGMSAGTTCTCAGCAGTTTCATACTGTTGC 1192 TCCGT L13:35GAAGGCAAGMSAGTTCTCCATAGTTTCATACTGTTGC 1193 TCCGT L13:36TGCGTAGAGGGCGTTCTCAAGGGAGAAACCTTGTTGT 1194 GTCAG L13:37CAGCAAGACGCAACCCAGAGTGTAGATCCCAACAGCT 1195 TGAAA L13:38CAGCAAGACGCAACCCAGAGTGTAGATCCCAACAGCT 1196 TGACC L13:39CAGCAAGACGCAACCCAGAGTGTAGATCCCAACAGCT 1197 TGCAT L13:40AGGTGTTTCCCTCTCCTCCTTAGCGACTTGCAGCTCT 1198 TGATC L13:41TTCGTAAGCTTGTCGAACCAGTGGCGGCATACTATCA 1199 GTAGT L13:42TTCGADAGCTTGTCGAACCAGTGGCGGCATACTATCA 1200 GTAGT L13:43GCCGAACAGGAAGGCTGGCTCTGCACCTTGGTGATCG 1201 TGGAG L13:44GCCGAACAGGAAGGCTGGCTCTGCACCTTGGTGATCT 1202 YTGAG L13:45ACACTTCAGCTGCTTCTCCAATCCACWTATGATCAGT 1203 TCAAG L13:46TGCCTTCAGCTGCTTCTCCAATCCACWTATGATCAGT 1204 TCAAG L13:47GCGCCAAGGTACCTTCTGCAGCTATCATTAAGACCCA 1205 CTTTC

The assembled library was then cloned into pVER7571. This vector is thesame as vector pVER7314 except for having a mutated ribosome bindingsite to reduce the amount of repressor produced per cell. Thismodification allows for more stringent assessment of repressor activityin the standard blue/white genetic plate assay, as well as in the liquidbased whole cell quantitative β-galactosidase assay. Following platingof the library, approximately 5,000 clones were re-arrayed and replicaplated onto M9 assay plates with no addition, or with 2 ppbethametsulfuron plus 0.002% arabinose. Colonies responding the strongestwith ethametsulfuron while remaining white without inducer were chosenas hits. One of the hits, L13-23, was found to be ˜3-fold improved overthe round 3 parent L7-A11 and to have the best repressor activity withinthis comparison (FIG. 11). Sequence changes of the round 5 hit comparedto parent molecule L7-A11 and wt TetR are shown in Table 20.

TABLE 20 Clone 55 60 64 67 68 82 86 88 100 104 105 108 113 116 121 134135 139 140 147 151 170 174 177 195 203 wt L L H F C N F C H R P K L Q CL S H F E H L I F C C L7-A11 M — A Y — — M — C G F Q A S — M Q I Y L L VL K — — L13-1-09 M — A Y — K M N A G F Q M S T F Q I Y L L V Y K — AL13-2-23 M F A Y — K M N A G F Q A C T F Q I Y L L V Y K — — L13-2-24 M— K Y L — M N C G F Q A W T F Q I Y L L V L H S A

Example 2 Plant Assay Development

A. Nicotiana benthamiana Leaf Infiltration Assay:

An in planta transient assay system was desired to rapidly confirmfunctionality of candidate SU-responsive repressors in planta prior totesting in transgenic plants. Therefore, an Agrobacterium based leafinfiltration assay was developed to measure repression and derepressionactivities. The strategy employed is to infiltrate leaves with a mixtureof reporter and effector (repressor) Agrobacterium strains such thatreporter activity is reduced by ˜90% in the presence of the effector andthen derepressed following treatment with inducer.

Two ethametsulfuron repressors, EsR A11 and EsR D01, were selected fortesting in conjunction with a wild type TetR control for dose responseto ethametsulfuron by transient expression in Nicotiana benthamianaleaves (FIG. 6). To this end, three test strains were derived bytransformation of Agrobacterium tumefaciens EHA105 with three differentT-DNA based vectors. Agrobacterium strains harboring binary vectors witha 35S::tetO-Renilla Luciferase reporter and dPCSV-tetR or -SuR effectorvariants were constructed. In addition to these tester cultures, anexisting Agrobacterium strain harboring a dMMV-GFP T-DNA was added tothe assay mixture to monitor the progression of Agrobacterium infectionfor sampling purposes.

To test the system for chemical switch activation, mixtures of testerAgrobacterium cultures containing 10% 35S::tetO-ReLuc reporter Agro, 10%dMMV-GFP Agro and 80% dPCSV-wt tetR Agro were infiltrated into N.benthamiana leaves and co-cultivated for 36 hours in the growth chamber.At this time the infiltrated leaves were excised and the petiole placedinto water (negative control) or inducer at the test concentrationsindicated in FIG. 6 and allowed to co-cultivate for another 36 hours.Infected leaf areas were assayed for Renilla luciferase activity andinducer treatments compared. The results show significant repression ofreporter activity (˜90%) with no inducer treatment (water control) forall tested repressors, and significant but incomplete induction of theEsR D01 repressor at inducer concentration as low as 0.02 ppm Es. BothEsR's were fully induced at 0.2 ppm Es whereas TetR was only fullyinduced at 2.0 ppm anhydrotetracycline (atc) (FIG. 6).

B. High Throughput in Planta Assay Development Using N. tabacum BY-2Cell Culture

In addition to the leaf assay it was desired to have an in planta assayto enable high throughput screening of SuR libraries for optimal plantfunctionality. We designed a system similar to the leaf assay but usingtobacco BY-2 cell culture in 96-well format. BY-2 cell culture wastransformed with a dMMV-HRA construct such that the culture wouldwithstand treatment with target sulfonylurea test compounds. Theresultant cell line grows and is fully resistant to 200 ppbchlorsulfuron.

Example 3 Operator Binding Assay

To confirm that sulfonylurea ligands were binding directly to themodified repressor molecules and causing derepression, an in vitro tetoperator gel shift study was undertaken.

An electrophoretic gel mobility shift assay (EMSA) of EsR variants wasdone to monitor binding to the tet operator (tetO) sequence and responseof the complex to inducers Es and Cs. TetO consists of a synthetic 48 bptetO-containing fragment created from hybridization of oligonucleotidetetO1 (SEQ ID NO: 1155):

5′-CCTAATTTTTGTTGACACTCTATCATTGATAGAGTTATTTTACCAC TC-3′and complementary oligonucleotide tetO2 (SEQ ID NO: 1156):

5′-GGATTAAAAACAACTGTGAGATAGTAACTATCTCAATAAAATGGTG AG-3′The tet operator is shown in bold.

An oligonucleotide and its complement of the same size containing nopalindromic sequence was used as a control (SEQ ID NO: 1157):

5′-CCTAATTTTTGTTGACTGTGTTAGTCCATAGCTGGTATTTTACCAC TC-3′and complementary oligonucleotide (SEQ ID NO: 1158):

5′-GGATTAAAAACAACTGACACAATCAGGTATCGACCATAAAATGGTG AG-3′

Five pmol of TetO or control DNA was mixed with the indicated amounts(FIG. 7) of ethametsulfuron repressor protein (L7A11) or BSA controlwith or without inducer in complex buffer containing 20 mM Tris-HCl(pH8.0) and 10 mM EDTA. The mixture was incubated at room temperaturefor 0.5 hour before loading onto the gel. The reaction waselectrophoresed on a Novex 6% DNA retardation gel (Invitrogen,EC6365BOX) at room temperature, 38 V in 0.5×TBE buffer for about 2hours. DNA was detected by ethidium bromide staining. The DNA sizemarker consists of the low DNA mass ladder (InVitrogen 10068-013).

The results are shown in FIG. 7. These results directly demonstrate thatthe modified repressors bind to operator DNA (lane 1 vs. lanes 3-5) andthen are released from the operator sequence in an inducer-specific anddose dependent manner. The data also indicate an inducer preference foroperator release by Es compared to Cs (lane 9 vs. 10). No change inoperator release could be detected by atc compared to no inducer (lane 5vs. 11).

Example 4 Binding and Dissociation Constants

Select SU repressors were further characterized for operator and ligandbinding, affinity and dissociation kinetics using Biacore™ SPRtechnology (Biacore, GE Healthcare, USA). The technology is based onsurface plasmon resonance (SPR), an optical phenomenon that enablesdetection of unlabeled interactants in real time. The SPR-basedbiosensors can be used in determination of active concentration,screening and characterization in terms of both affinity and kinetics.

The kinetics of an interaction, i.e., the rates of complex formation(k_(a)) and dissociation (k_(d)), can be determined from the informationin a sensorgram. If binding occurs as sample passes over a preparedsensor surface, the response in the sensorgram increases. If equilibriumis reached, a constant signal is seen. Replacing the sample with buffercauses the bound molecules to dissociate and the response decreases.Biacore evaluation software generates the values of k_(a) and k_(d) byfitting the data to interaction models.

The affinity of an interaction is determined from the level of bindingat equilibrium (seen as a constant signal) as a function of sampleconcentration. Affinity can also be determined from kineticmeasurements. For a simple 1:1 interaction, the equilibrium constantK_(D) is the ratio of the kinetic rate constants, k_(d)/k_(a).

A. Operator Binding Characterization of Repressors

Repressor k_(a) (M⁻¹ s⁻¹) K_(d) (s⁻¹) K_(D) (nM) Wt TetR 3.3 × 10⁵ 3.0 ×10⁻³  9.0 ± 1.0 L7-1C03-A5 4.7 × 10⁴ 7.8 × 10⁻³ 150 ± 5 L7-3E03-D1 5.5 ×10⁴ 1.1 × 10⁻² 200 ± 50 L7-1F08-A11 7.1 × 10⁴ 1.7 × 10⁻² 250 ± 120L7-1G06-B2 4.6 × 10⁴ 1.9 × 10⁻² 430 ± 160B. SU Binding Characterization of Repressors

KD (M) ATC + Repressor Es + Mg Es − Mg Cs + Mg Cs − Mg Mg L7-1C03-A50.46 1.78 83 365 Null L7-1F08-A11 0.45 1.09 40 92 Null L7-1G06-B2 0.532.15 60 255 Null L7-3E03-D1 0.73 2.15 48 115 Null Wt TetR Null Null NullNull 0.0036

Example 5 Sulfonyl Urea Repressor Ligand-Binding Domain Fusions

The ligand binding domains from the sulfonylurea repressors providedherein can be fused to alternative DNA binding domains in order tocreate further sulfonylurea repressors that selectively and specificallybind to other DNA sequences (e.g., Wharton and Ptashne (1985) Nature316:601-605). Many domain swapping experiments have been published,demonstrating the breadth and flexibility of this approach. Generally,an operator binding domain or specific amino acid/operator contactresidues from a different repressor system will be used, but other DNAbinding domains can also be used. For example, a polynucleotide encodinga TetR(D)/SuR chimeric polypeptide consisting of the DNA binding domainfrom TetR(D) (e.g., amino acid residues 1-50) and ligand binding domainof a SuR residues (e.g., amino acid residues 51-208 from TetR(B) can beconstructed using any standard molecular biology method or combinationthereof, including restriction enzyme digestion and ligation, PCR,synthetic oligonucleotides, mutagenesis or recombinational cloning. Forexample, a polynucleotide encoding a SuR comprising a TetR(D)/SuRchimera can be constructed by PCR (Landt et al. (1990) Gene 96:125-128;Schnappinger et al. (1998) EMBO J 17:535-543) and cloned into a suitableexpression cassette and vector. Any other TetOp binding domains can besubstituted to produce a SuR that specifically binds to the cognate tetoperator sequence.

In addition, mutant TetO^(c) binding domains from variant TetR's havingsuppressor activity on constitutive operator sequences (tetO-4C andtetO-6C) can be used (see, e.g., Helbl and Hillen (1998) J Mol Biol276:313-318; and Helbl et al. (1998) J Mol Biol 276:319-324). Further,the polynucleotides encoding these DNA binding domains can be modifiedto change their operator binding properties. For example, thepolynucleotides can be shuffled to enhance the binding strength orspecificity to a wild type or modified tet operator sequence, or toselect for specific binding to a new operator sequence.

Additional variants could be made by fusing an SuR repressor, or an SuRligand binding domain to an activation domain. Such systems have beendeveloped using Tet repressors. For example, one system converted a tetrepressor to an activator via fusion of the repressor to atranscriptional transactivation domain such as herpes simplex virus VP16and the tet repressor (tTA, Gossen and Bujard (1992) Proc Natl Acad SciUSA 89:5547-5551). The repressor fusion is used in conjunction with aminimal promoter which is activated in the absence of tetracycline bybinding of tTA to tet operator sequences. Tetracycline inactivates thetransactivator and inhibits transcription.

Example 6 Testing of Repressor Proteins in Soybean

Any transformation protocols, culture techniques, soybean source, andmedia, and molecular cloning techniques can be used with thecompositions and methods.

A Transformation and Regeneration of Soybean (Glycine max)

Transgenic soybean lines are generated by the method of particle gunbombardment (Klein et al. Nature 327:70-73 (1987); U.S. Pat. No.4,945,050) using a BIORAD Biolistic PDS1000/He instrument and eitherplasmid or fragment DNA. The following stock solutions and media areused for transformation and regeneration of soybean plants:

Stock Solutions:

-   Sulfate 100× Stock: 37.0 g MgSO4.7H2O, 1.69 g MnSO4.H2O, 0.86 g    ZnSO4.7H2O, 0.0025 g CuSO4.5H2O-   Halides 100× Stock: 30.0 g CaCl2.2H2O, 0.083 g KI, 0.0025 g    CoCl2.6H2O-   P, B, Mo 100× Stock: 18.5 g KH2PO4, 0.62 g H3BO3, 0.025 g    Na2MoO4.2H2O-   Fe EDTA 100× Stock: 3.724 g Na2EDTA, 2.784 g FeSO4.7H2O-   2,4-D Stock: 10 mg/mL 2,4-Dichlorophenoxyacetic acid-   B5 vitamins, 1000× Stock: 100.0 g myo-inositol, 1.0 g nicotinic    acid, 1.0 g pyridoxine HCl, 10 g thiamine.HCL.

Media (Per Liter):

-   SB199 Solid Medium: 1 package MS salts (Gibco/BRL, Cat. No.    11117-066), 1 mL B5 vitamins 1000× stock, 30 g Sucrose, 4 ml 2,4-D    (40 mg/L final concentration), pH 7.0, 2 g Gelrite-   SB1 Solid Medium: 1 package MS salts (Gibco/BRL, Cat. No.    11117-066), 1 mL B5 vitamins 1000× stock, 31.5 g Glucose, 2 mL 2,4-D    (20 mg/L final concentration), pH 5.7, 8 g TC agar-   SB196: 10 mL of each of the above stock solutions 1-4, 1 mL B5    Vitamin stock, 0.463 g (NH4)2SO4, 2.83 g KNO3, 1 mL 2,4 D stock, 1 g    asparagine, 10 g sucrose, pH 5.7-   SB71-4: Gamborg's B5 salts, 20 g sucrose, 5 g TC agar, pH 5.7.-   SB103: 1 pk. Murashige & Skoog salts mixture, 1 mL B5 Vitamin stock,    750 mg MgCl2 hexahydrate, 60 g maltose, 2 g gelrite, pH 5.7.-   SB166: SB103 supplemented with 5 g per liter activated charcoal.    Soybean Embryogenic Suspension Culture Initiation:

Soybean cultures are initiated twice each month with 5-7 days betweeneach initiation. Pods with immature seeds from available soybean plants45-55 days after planting are picked, removed from their shells andplaced into a sterilized magenta box. The soybean seeds are sterilizedby shaking them for 15 min in a 5% Clorox solution with 1 drop of ivorysoap (i.e., 95 mL of autoclaved distilled water plus 5 mL Clorox and 1drop of soap, mixed well). Seeds are rinsed using 2 1-liter bottles ofsterile distilled water and those less than 3 mm are placed onindividual microscope slides. The small end of the seed is cut and thecotyledons pressed out of the seed coat. Cotyledons are transferred toplates containing SB199 medium (25-30 cotyledons per plate) for 2 weeks,then transferred to SB1 for 2-4 weeks. Plates are wrapped with fibertape. After this time, secondary embryos are cut and placed into SB196liquid media for 7 days.

Culture Conditions:

Soybean embryogenic suspension cultures (cv. Jack) are maintained in 50mL liquid medium SB196 on a rotary shaker, 150 rpm, 26° C. with coolwhite fluorescent lights on 16:8 h day/night photoperiod at lightintensity of 60-85 μE/m2/s. Cultures are subcultured every 7 days to twoweeks by inoculating approximately 35 mg of tissue into 50 mL of freshliquid SB196 (the preferred subculture interval is every 7 days).

Preparation of DNA for Bombardment:

In particle gun bombardment procedures it is possible to use purifiedintact plasmid DNA; or DNA fragments containing only the recombinant DNAexpression cassette(s) of interest. For every seventeen bombardmenttransformations, 85 μL of suspension is prepared containing 1 to 90picograms (pg) of plasmid DNA per base pair of each DNA plasmid. Bothrecombinant DNA plasmids are co-precipitated onto gold particles asfollows. The DNAs in suspension are added to 50 μL of a 10-60 mg/mL 0.6μm gold particle suspension and then combined with 50 μL CaCl2 (2.5 M)and 20 μL spermidine (0.1 M). The mixture is vortexed for 5 sec, spun ina microfuge for 5 sec, and the supernatant removed. The DNA coatedparticles are then washed once with 150 μL of 100% ethanol, vortexed andpelleted, then resuspended in 85 μL of anhydrous ethanol. Five μL of theDNA coated gold particles are then loaded on each macrocarrier disk.

Tissue Preparation and Bombardment with DNA:

Approximately 150 to 250 mg of two-week-old suspension culture is placedin an empty 60 mm×15 mm petri plate and the residual liquid removed fromthe tissue using a pipette. The tissue is placed about 3.5 inches awayfrom the retaining screen and each plate of tissue is bombarded once.Membrane rupture pressure is set at 650 psi and the chamber is evacuatedto −28 inches of Hg. Following bombardment, the tissue from each plateis divided between two flasks, placed back into liquid media, andcultured as described above.

Selection of Transformed Embryos and Plant Regeneration:

After bombardment, tissue from each bombarded plate is divided andplaced into two flasks of SB196 liquid culture maintenance medium perplate of bombarded tissue. Seven days post bombardment, the liquidmedium in each flask is replaced with fresh SB196 culture maintenancemedium supplemented with 100 ng/ml selective agent (selection medium).Transformed soybean cells can be selected using a sulfonylurea (SU)compound such as 2 chloro N ((4 methoxy 6 methy 1,3,5 triazine 2yl)aminocarbonyl)benzenesulfonamide (common names: DPX-W4189 andchlorsulfuron). Chlorsulfuron (Cs) is the active ingredient in theDuPont sulfonylurea herbicide, GLEAN®. The selection medium containingSU is replaced every two weeks for 6-8 weeks. After the 6-8 weekselection period, islands of green, transformed tissue are observedgrowing from untransformed, necrotic embryogenic clusters. Theseputative transgenic events are isolated and kept in SB196 liquid mediumwith Cs at 100 ng/ml for another 2-6 weeks with media changes every 1-2weeks to generate new, clonally propagated, transformed embryogenicsuspension cultures. Embryos spend a total of around 8-12 weeks incontact with Cs. Suspension cultures are subcultured and maintained asclusters of immature embryos and also regenerated into whole plants bymaturation and germination of individual somatic embryos.

Somatic embryos became suitable for germination after four weeks onmaturation medium (1 week on SB166 followed by 3 weeks on SB103). Theyare then removed from the maturation medium and dried in empty petridishes for up to seven days. The dried embryos are then planted in SB714 medium where they are allowed to germinate under the same light andtemperature conditions as described above. Germinated embryos aretransferred to potting medium and grown to maturity for seed production.

B. Vector Construction and Testing

Plasmids were made using standard procedures and from these plasmids DNAfragments were isolated using restriction endonucleases and agarose gelpurification according to the protocol described in Example 6A. Each DNAfragment contained three cassettes. Cassette 1 is a reporter expressioncassette; Cassette 2 is the repressor expression cassette; and, Cassette3 is an expression cassette providing an HRA gene. The repressors testedin Cassette 2 are described in Table 21. The polynucleotides comprisingthe repressor coding region were synthesized to comprise plant preferredcodons. In all cases Cassette 1 contained a 35S cauliflower mosaic viruspromoter having three tet operators introduced near the TATA box (Gatzet al. (1992) Plant J 2:397-404 (3XOpT 35S)) driving expression of DsRedfollowed by the 35S cauliflower mosaic virus 3′ terminator region. Inall cases cassette three contained the S-adenosylmethionine synthasepromoter followed by the HRA version of the acetolactase synthase (ALS)gene followed by the Glycine max ALS 3′ terminator. The HRA version ofthe ALS gene confers resistance to sulfonylurea herbicides. EF1A1 is thepromoter of a soybean translation elongation factor EF1 alpha describedin patent publication US20080313776.

TABLE 21 Fragment Fragment Repressor Repressor Fragment Name aliasCassette 2 alias SEQ ID SEQ ID PHP37586A CHSW004 EF1A1::EsR1::Nos3′L7-IC3-A5 1240 1222 PHP37587A CHSW005 EF1A1::EsR2::Nos3′ L7-1F8-A11 12411223 PHP37588A CHSW006 EF1A1::EsR2::Nos3 L7-1G6-B2 1242 1224 PHP37589ACHSW007 EF1A1::EsR4::Nos3′ L7-3E3-D1 1243 1225 PHP39389A CHSW010EF1A1::EsR5::CaMV35S3′ L12-1-10 1232 1226 PHP39390A CHSW011EF1A1::EsR6::CaMV35S3′ L13-2-23 1233 1227

DNA fragments were used for soybean transformation according to theprotocol described in Example 6A. Plants were regenerated and leaf discs(˜0.5 cm) were harvested from young leaves. The leaf discs wereincubated in SB103 liquid media containing 0 ppm, 0.5 ppm or 5 ppmethametsulfuron for 2-5 days. Ethametsulfuron (product number PS-2183)was purchased from Chem Service (West Chester, Pa.) and solubilized ineither 10 mM NaOH or 10 mM NH₄OH. On each day leaf discs were examinedunder a dissecting microscope with a DsRed band pass filter. Thepresence of DsRed was scored visually.

Plants that expressed DsRed at 0 time were scored as leaky. Plants thatdid not express DsRed after five days were scored as negative. Plantsthat expressed DsRed after addition of ethametsulfuron were scored asinducible. Results from plants derived from DNAs described in Table 21are shown in Table 22.

TABLE 22 Total % % % Name Alias Events Leaky Negative InduciblePHP37586A CHSW004 12 33 33 33 PHP37587A CHSW005 28 7 50 43 PHP37588ACHSW006 6 0 0 100 PHP37589A CHSW007 9 0 22 78 PHP39389A CHSW010* 19 5 2642 PHP39390A CHSW011* 35 0 17 57 *In these cases the total does notequal 100% as multiple plants were examined from some events and, insome cases, different plants from the same event behaved differently.

This shows that the repressor protein responds to ethametsulfuron byinducing expression of DsRed. Plants derived from the first fourfragments showed visual evidence of DsRed after three days ofincubation. Plants derived from the last two fragments showed visualevidence of DsRed after two days of incubation. The presence of DsRedwas scored visually, but this was confirmed by Western Blot analysis ona selection of transformants using a rabbit polyclonal antibody(ab41336) from Abcam (Cambridge, Mass.).

Leaf punches were harvested as described above from a selection oftransformants and incubated in SB103 media with 0, 5, 50, 250 and 500ppb ethametsulfuron. At all concentrations of ethametsulfuron, leavesshowed visual evidence of DsRed after three days of incubation. At thelowest concentration (5 ppb) the presence of DsRed was limited to a“halo” near the outside edge of the leaf disc.

Plants were allowed to mature as described in Example 6A. Since soybeansare self fertilizing, the T1 seeds derived from these plants would beexpected to segregate 1 wild type: 2 hemizyogote: 1 homozygote if onlyone transgene locus was created during transformation. Sixteen seedsfrom five different events were planted and allowed to germinate. Leafpunches were collected from young seedlings and incubated in SB103 mediawith 0 and 5 ppm ethametsulfuron. Leaf discs were scored for DsRedexpression and 0 and 3 days and results are shown in Table 23.

TABLE 23 Total # # # # In- Seeds Leaky Negative ducible Name Event IDGerminated Plants Plants Plants PHP37586A 6048.3.8.3 11 0 2 9 PHP37587A6049.2.2.4 12 0 5 7 PHP37588A 6150.3.2.1 14 0 1 13 PHP37589A 6154.4.5.115 0 15 0 PHP39389A 6151.4.18.1 12 3 9 0

Example 7 Testing of Repressor Proteins in Corn

To evaluate SU repressors in plants, RFP reporter constructs wereconstructed and transformed into maize cells via Agrobacterium using thefollowing T-DNA configuration:

RB-35S/TripleOp/Pro::RFP-Ubi Pro::EsR-HRA cassette-PAT cassette-LB.

Using standard molecular biology and cloning techniques, T-DNA vectorshaving the configuration above comprising selected round 3 SU repressors(EsRs) were constructed. The polynucleotides comprising the repressorcoding region were synthesized to comprise plant preferred codons. Theconstructs are summarized below:

SU repressor alias SU repressor Construct ID (EsR) SEQ ID PHP37707L7-1C3-A5 1240 PHP37708 L7-1F8-A11 1241 PHP37709 L7-1G6-B2 1242 PHP37710L7-3E3-D1 1243

The reporter construct T-DNA contained a 35S promoter with two tetoperators flanking the TATA box and one downstream adjacent to thetranscription start site (as described by Gatz et al. (1992) Plant Cell2:397-404) driving expression of the Red Fluorescent Protein gene, aubiquitin driven SU repressor (EsR), an expression cassette containingthe maize HRA gene for SU resistance and a moPAT expression cassette forselection.

Immature embryos were transformed using standard methods and media.Briefly, immature embryos were isolated from maize and contacted with asuspension of Agrobacterium, to transfer the T-DNA's containing thesulfonylurea expression cassette to at least one cell of at least one ofthe immature embryos. The immature embryos were immersed in anAgrobacterium suspension for the initiation of inoculation and culturedfor seven days. The embryos were then transferred to culture mediumcontaining carbinicillin to kill off any remaining Agrobacterium. Next,inoculated embryos were cultured on medium containing both carbinicillinand bialaphos (a selective agent) and growing transformed callus wasrecovered. The callus was then regenerated into plantlets on solid mediabefore transferring to soil to produce mature plants. Approximately 10single copy events from each of the constructs were sent to thegreenhouse.

To evaluate de-repression, callus was transferred to medium with andwithout ethametsulfuron and chlorsulfuron and RFP Fluorescence wasobserved under the microscope (see FIG. 10A). Most events de-repressedand there were no obvious differences between the round three repressorstested. To evaluate de-repression in plants, seeds for single copyplants were germinated in the presence of ethamethsulfron andfluorescence was observed and photographed (see FIG. 10B). As a positivecontrol, a vector containing the same configuration of expressioncassettes as PHP37707-10, but with UBI::TetR in place of UBI::EsR, weretransformed into maize immature embryos and tested for induction ondoxycline. When grown in the presence of 1 mg/l doxycycline, transgeniccallus and plants containing the TetR expression cassette induced over asimilar 5-6 day period.

The articles “a” and “an” refer to one or more than one of thegrammatical object of the article. By way of example, “an element” meansone or more of the element. All book, journal, patent publications andgrants mentioned in the specification are indicative of the level ofthose skilled in the art. All publications and patent applications areherein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference. Although theforegoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding,certain changes and modifications may be practiced within the scope ofthe appended claims. These examples and descriptions are illustrativeand are not read as limiting the scope of the appended claims.

What is claimed:
 1. An isolated polypeptide comprising asulfonylurea-responsive repressor that specifically binds to apolynucleotide comprising an operator sequence, wherein the binding isregulated by a sulfonylurea compound, and wherein said polypeptidecomprises an amino acid sequence having at least 90% sequence identityto SEQ ID NO:1233.
 2. The polypeptide of claim 1, wherein saidpolypeptide comprises an amino acid sequence having at least 95%sequence identity to SEQ ID NO:1233.